Biomarkers in Peripheral Blood Mononuclear Cells for Diagnosing or Detecting Lung Cancers

ABSTRACT

Methods and compositions are provided for diagnosing or detecting a condition, e.g., lung disease in a mammalian subject by use of a micro-RNA expression level or an expression level profile of multiple miRNA in the peripheral blood mononuclear cells (PBMC) of the subject which is characteristic of COPD or NSCLC. Detection of changes in expression in specific miRNA biomarkers from that of a reference sample or miRNA expression profile are correlated with non-small cell lung cancer (NSCLC) and/or COPD and permit differentiation among healthy subjects, subjects with COPD and subjects with adenocarcinoma or squamous cell carcinoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/719,826, filed May 22, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/127,161, filed May 2, 2011, now U.S. Pat. No.9,068,974, which is a national stage of International Patent ApplicationNo. PCT/US2009/063603, filed Nov. 6, 2009, which claims the benefit ofthe priority of U.S. Provisional Patent Application No. 61/112,744,filed Nov. 8, 2008 (expired), which applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Lung cancer is the most common worldwide cause of cancer mortality.Non-small cell lung cancer (NSCLC) is a highly lethal disease with cureonly possible by early detection followed by surgery. Unfortunately, atthe time of diagnosis only 15% of patients with lung cancer havelocalized disease. Field cancerization in which the lung epitheliumbecomes mutagenized following exposure to cigarette smoke makes itdifficult to identify genetic changes that differentiate smokers fromsmokers with early lung cancer.

One of the most important long-term goals in improving lung cancersurvival is to detect malignant tumors in high-risk patients, primarilysmokers and former smokers, who represent the majority of all lungcancer cases, at an early stage, while they are still surgicallyresectable. Although most patients with lung cancer are smokers orex-smokers, the actual incidence of lung cancer in this group is quitesmall (<0.2%). Various studies have demonstrated that the incidence oflung cancer in male cigarette smokers ranged from 0.16% (in Caucasians)to 0.26% (in African Americans). Another high-risk population issubjects with lung nodules of unknown etiology, as identified onscreening chest X-rays, computerized axial tomography (CT) scans, lowdose spiral CT scans, or incidentally. Currently, the only way todifferentiate benign from malignant nodules is an invasive biopsy,surgery, or prolonged observation with repeated scanning.

While these methods may identify non-calcified pulmonary nodules inapproximately 30% of screened smokers and ex-smokers (with a range of13-51% of patients on prevalence screen; 2-13% on annual screen), only asmall number are found to be lung cancers (0.4 to 2.7%). Thus, about 3to 12% of subjects with detected non-calcified nodules prompt aninvasive diagnostic workup. The high false positive rate of CT scanningrequires patients to undergo extensive follow-up investigations withserial CT, positron emission tomography (PET scan), and/or biopsy.Studies indicate that 20-55% of patients undergoing surgical lung biopsyfor indeterminate lung nodules are subsequently found to have benigndisease.

One established and validated method to achieve the goal of geneticdiagnosis has been the use of microarray signatures from tumor tissue.Peripheral blood mononuclear cells (PBMC) profiles can be used todiagnose and classify systemic diseases, including cancer, and tomonitor therapeutic response. The validity of using PBMC gene expressionprofiles in patients with cancer has been previously reported in the useof microarrays to compare PBMC from patients with late stage renal cellcarcinoma compared to normal controls. A 37 gene classifier has beendeveloped for detecting early breast cancer from peripheral bloodsamples with 82% accuracy. Another study identified gene expressionprofiles in the PBMC of colorectal cancer patients that could becorrelated with response to therapy.

While the effect of chronic obstruction pulmonary disease (COPD) on PBMCgene expression is relatively unstudied to date, there are some limitedreports about the effect of cigarette smoke. Exposure of peripheralblood lymphocytes (PBL) ex vivo to cigarette smoke induced many changesin gene expression. Changes could be detected in the transcriptosome ofblood neutrophils in COPD patients versus normals. Gene expression inairway epithelia of smokers, ex-smokers and non-smokers has beencompared. Although many clinical manifestations of smoking rapidlyreturned to normal after smoking cessation, there was a subset of geneswhose expression remained altered.

MicroRNAs (miRNAs) are a large group of ribonucleic acid sequences,isolated and identified from insects, microorganisms, humans, animalsand plants, which are reported in databases including that of TheWellcome Trust Sanger Institute (http://miRNA.sanger.ac.uk/sequences/).These miRNAs are about 22 nucleotides in length and arise from longerprecursors, which are transcribed from non-protein-encoding genes. Theprecursors form structures that fold back on themselves inself-complementary regions. Relatively little is known about thefunctional role of miRNAs and even less on their targets. It is believedthat miRNA molecules interrupt or suppress gene translation throughprecise or imprecise base-pairing with their targets (US PublishedPatent Application No. 2004/0175732). Bioinformatics analyses suggestthat any given miRNA may bind to and alter the expression of up toseveral hundred different genes; and a single gene may be regulated byseveral miRNAs. The complicated interactive regulatory networks amongmiRNAs and target genes have been noted to make it difficult toaccurately predict which genes will actually be misregulated in responseto a given miRNA. Expression levels of certain miRNAs have beenassociated with various cancers (Esquela-Kerscher and Slack, 2006 Nat.Rev. Cancer, 6(4):259-269; McManus 2003 Seminars in Cancer Biology,13:253-258; Karube Y et al 2005 Cancer Sci, 96(2):111-5; Yanaihara N. etal 2006 Cancer Cell, 9(3):189-98).

There remains a need in the art for new and effective tools tofacilitate early diagnoses of various lung cancers, as well as lessinvasive diagnostic tests that could more accurately diagnose malignantdisease in patients from other non-malignant diseases and would reduceunnecessary diagnostic surgery, biopsies, PET scans, and/or repeated CTscans.

SUMMARY OF THE INVENTION

In one embodiment, a method of diagnosing or detecting, detecting orassessing a condition in a mammalian subject is provided. This methodcomprises detecting in a biological sample of the subject, or from anmiRNA expression profile generated from the sample, the expression levelof an miRNA nucleic acid sequence identified in Table 1. The miRNAexpression level or miRNA expression profile in the subject's sample iscompared to a reference miRNA standard. A change in expression of thesubject's sample miRNA from that in the reference miRNA standardindicates a diagnosis, detection, discrimination or prognosis of aselected condition. The condition detected includes a lung cancer, suchas a type or stage of a non-small cell lung cancer, chronic obstructivepulmonary disease (COPD), and benign lung nodules. In certainembodiments, the condition detected or discriminated is a lung cancer ina subject post surgical resection of a tumor. This condition can bemonitored by comparing an miRNA or miRNA profile or pattern obtainedbefore surgery with the post-surgical levels of the miRNA. In certainembodiments, the biological sample is whole blood, peripheral bloodmononuclear cells, plasma or serum.

In another aspect, a method of diagnosing or detecting or assessing alung disease in a mammalian subject is provided, comprising detecting ina sample of the subject's peripheral blood mononuclear cells (PBMC), orfrom an miRNA expression profile generated from the sample, theexpression level of at least one of the miRNA nucleic acid sequences ofTable 1. In another embodiment, the same method is performed using adifferent sample, e.g., whole blood. The miRNA expression level of thespecified miRNA in the sample is compared to an average or standardexpression level of the same miRNA in a reference sample or profile. Achange in expression of at least one of these miRNA from that in thereference indicates a diagnosis of non small cell lung cancer (NSCLC) orchronic obstructive pulmonary disease (COPD). In one embodiment theNSCLC is adenocarcinoma or squamous cell carcinoma. In anotherembodiment the NSCLC is early stage (I or II) adenocarcinoma or earlystage (I or II) squamous cell carcinoma. In another embodiment, thechange in expression of the miRNA is useful to monitor patients who havehad a cancerous lung tumor surgically removed by comparing the miRNAwith the same miRNA pre-surgery. In certain embodiments of thesemethods, the reference standard utilized is a standard or profilederived from the same sample, e.g., PBMC or whole blood, of a referencehuman subject or an average of multiple subjects with a particularphysical condition, as defined herein.

In another aspect, a method of diagnosing or detecting a lung disease orcondition in a mammalian subject is provided, comprising detecting in asample of the subject's peripheral blood mononuclear cells (PBMC), orfrom an miRNA expression profile generated from the sample, theexpression level of at least one of the miRNA nucleic acid sequenceshsa-miR-148a, hsa-miR-142-5p, hsa-miR-221, hsa-miR-let-7d, has-miR-328,hsa-miR-let-7a, hsa-miR-let-7c, hsa-miR-34a, hsa-miR-202,hsa-miR-769-5p, and hsa-miR-642. In another embodiment, the same methodis performed using a different sample, e.g., whole blood. The miRNAexpression level of the specified miRNA in the sample is compared to anaverage or standard expression level of the same miRNA in a referencesample or profile. A change in expression of at least one of these miRNAfrom that in the reference is indicative of a non small cell lung cancer(NSCLC) or chronic obstructive pulmonary disease (COPD). In oneembodiment the NSCLC is adenocarcinoma or squamous cell carcinoma. Inanother embodiment the NSCLC is early stage (I or II) adenocarcinoma orearly stage (I or II) squamous cell carcinoma. In certain embodiments ofthese methods, the reference standard utilized is a standard or profilederived from same sample, e.g., the PBMC or whole blood, of a referencehuman subject or an average of multiple subjects with a particularphysical condition, as defined below.

In another aspect, methods for diagnosing or detecting adenocarcinomaare accomplished by detecting in a sample of a subject's PBMC changes incertain miRNA expression levels or miRNA expression profiles asidentified below from those of a reference standard or profile. Inanother embodiment, the same method is performed using a differentsample, e.g., whole blood.

In another aspect, methods for diagnosing or detecting squamous cellcarcinoma are accomplished by detecting in a sample of a subject's PBMCchanges in certain miRNA expression levels or miRNA expression profilesas identified below from those of a reference standard or profile. Inanother embodiment, the same method is performed using a differentsample, e.g., whole blood.

In another aspect, methods for diagnosing or detecting chronicobstructive pulmonary disease are accomplished by detecting in a sampleof a subject's PBMC changes in certain miRNA expression levels or miRNAexpression profiles as identified below from those of a referencestandard or profile. In another embodiment, the same method is performedusing a different sample, e.g., whole blood.

In another aspect, a composition, diagnostic reagent, kit, or microarrayfor diagnosing or detecting a condition, e.g., a lung disease, in amammalian subject employs at least two ligands, e.g., nucleic acidprimers, wherein each ligand hybridizes to or complexes with oridentifies a single different miRNA nucleic acid sequence from among twoor more miRNAs identified in Table 1 and FIG. 3 as characteristic of aparticular condition, e.g., lung disease. In certain embodiments, theprimers are immobilized on a substrate.

In another aspect, a composition, kit, or microarray for diagnosing ordetecting a lung disease in a mammalian subject contains at least twonucleic acid primers, wherein each the primer hybridizes with a singledifferent miRNA nucleic acid sequence. The miRNA are characteristic of aparticular condition, e.g., lung disease and include one or more miRNAselected from: hsa-miR-148a, hsa-miR-142-5p, hsa-miR-221,hsa-miR-let-7d, hsa-miR-let-7a, hsa-miR-328, hsa-miR-let-7c,hsa-miR-34a, hsa-miR-202, hsa-miR-769-5p, hsa-miR-642.

In another aspect, the composition, kit, or microarray employsantibodies, fragments or probes that identify one or more of the miRNAsdisclosed herein.

The methods and compositions described herein allow the physician todistinguish between non-cancerous conditions, such as COPD or benignlung nodules, from lung cancer, such as NSCLC. The methods andcompositions described herein allow the physician to monitor the statusof a lung cancer patient post-surgical resection of a tumor.

Other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows TaqMan® Low Density Array (TLDA) reproducibility andsensitivity based on correlation analyses of miRNA expression in threetechnical replicates of an early stage adenocarcinoma (AC-E) sample.This graph shows Spearman correlation (r) between the replicates basedon cycle threshold (Ct) less than 39.

FIG. 1B shows a TLDA reproducibility and sensitivity based on the samecorrelation analyses of miRNA expression as in FIG. 1A. This graphrepresents the results of sliding correlation analysis. Calculation ofaverage Spearman correlation is based on expression of 40 miRNAs with Ctvalues between 28 and 37.5. Ct threshold is the maximum Ct value among 3replicates.

FIG. 2 is a graph showing the effect of higher input RNA on miRNAdetectability by TLDA: comparison of 50 and 100 ng of early stagesquamous cell carcinoma (SCC) pooled peripheral blood mononuclear cellssamples. Each dot represents a newly detected miRNA that was expressedin 50 ng sample at the Ct value listed on X-axis. All graphed miRNAs areexpressed below cycle 31 in 100 ng sample. One-half cycle difference istaken as technical error by default. Linear regression analysis showshigh correlation of initial Ct values detected in 50 ng sample withnormalized cycle difference calculated using both 50 and 100 ng sampledata.

FIG. 3 is a table of data obtained from a hierarchical clustering of 43peripheral blood mononuclear cells-derived miRNAs differentiallyexpressed in RNA pools. Each listed miRNA is identified by name, asreported in Table 1, and was expressed at Ct≤31 and had about 2-folddifference between any pair of pooled peripheral blood mononuclear cellssamples. Hierarchical clustering (not shown) was done using normalizedEuclidean distance for samples and correlation similarity metric formiRNAs. The data from the clustering are reported in the Figure usingarbitrary expression units under each subject population. COPDrepresents a population having chronic obstructive pulmonary disease(predominantly former smokers); AC-E represents early stage (stage I+II)adenocarcinoma; SCC-E represents early stage I and II squamous cellcarcinoma; S represents a population of smokers without disease; NSrepresents a population of healthy subjects who have never smoked. “Maxratio” represents the fold difference in expression between the twogroups furthest apart in expression, adjacent the identity of the twogroups. The final column is the ratio between expression in COPD vs.expression in all NSCLC subjects. For miRNA expression, a difference of1.2 fold and above is considered significant between two groups.Negative numbers are indicative of upregulation of the miRNA in theNSCLC subjects compared to COPD. Positive numbers are indicative ofdownregulation of the miRNA in the NSCLC subjects compared to COPDpatients. Each PBMC-derived miRNA was differentially expressed in 5 RNApools. Each miRNA was expressed at Ct≤31 and had at least 2-folddifference between any pair of pooled PBMC samples. Hierarchicalclustering was done using normalized Euclidean distance for samples andcorrelation similarity metric for miRNAs (not shown).

FIG. 4A is an expression profiles for miR-148a of TLDA-derivedperipheral blood mononuclear cells miRNAs run on 10 new individualpatient samples using single TaqMan® miRNA assays. TLDA-derivednormalized expression is represented by (●); COPD represents apopulation having chronic obstructive pulmonary disease (predominantlyformer smokers); AC-E represents early stage (stage I+II)adenocarcinoma; SCC-E represents early stage I and II squamous cellcarcinoma; S represents smokers without disease; NS represents healthysubjects who have never smoked; (*) represents p<0.05; and (**)represents p<0.01.

FIG. 4B is an expression profile for miR-142-5p for the samples andusing the symbols as defined in FIG. 4A.

FIG. 4C is an expression profile for miR-221 for the samples and usingthe symbols as defined in FIG. 4A.

FIG. 4D is an expression profile for miR-let-7d for the samples andusing the symbols as defined in FIG. 4A.

FIG. 5 is a bar graph showing that the expression of endogenous controlson TLDA is consistent within every sample tested and can be used fornormalization. The graph plots RNU44 and RNU48 absolute expressionvalues (AE) across 6 pooled PBMC samples (50 ng). AC-E sample was run on3 different TLDAs and thus represented 3 times; LC (late stage III andIV) lung cancer sample was run as a positive control.

FIG. 6A is a Spearman correlation analyses of AC-E samples when 48-plexand mega-plex RT primers were used during reverse transcription step.Data was normalized using average expression of RNU44 and RNU48. miRNAexpression profiles were detected using 8 RT primer pools (48-plex) and1 RT primer pool (450-plex) protocols, and are highly correlated.

FIG. 6B is a Spearman correlation analyses of SCC-E samples when 48-plexand mega-plex RT primers were used during reverse transcription step, asdescribed for FIG. 6A.

FIG. 7A is a Spearman correlation analyses of AC-E samples when miRNAexpression was compared for only mega-plex (450 miRNA) RT pool with andwithout preamplification. Data was normalized using average expressionof RNU44 and RNU48. Pre-amplification procedure substantially increasedthe number of detected miRNAs but did not affect overall miRNAexpression.

FIG. 7B is a Spearman correlation analyses of SCC-E samples using thesame procedure as described for FIG. 7A.

FIG. 8 is a bar graph showing pre-surgery (PRE)/post-surgery (POST)ratios for miRNAs with false discovery rate (FDR)<15%. The data ispresented in log scale to accent patients where miRNAs did not showupregulation. The numbers indicate the specific patient. Black squarerepresents miR-let-7c; white square represents miR-34a; gray squarerepresents mIR-202*, white cross-hatched square represents mIR-769-5pand dark cross-hatched square represents miR-642.

DETAILED DESCRIPTION OF THE INVENTION

The methods and compositions described herein apply miRNA expressiontechnology to blood screening for the detection, diagnosis, andmonitoring of response to treatment of a condition, such as a lungdisease. In certain embodiments, the lung disease is an NSCLC or COPD.The compositions and methods described herein permit the diagnosis ordetection of a condition or disease or its stage generally, and lungcancers and COPD particularly, by determining changes in acharacteristic miRNA or miRNA expression profile derived from abiological sample, including in various embodiments, whole blood,peripheral blood mononuclear cells (PBMC) or peripheral bloodlymphocytes (PBL) of a mammalian, preferably human, subject. The miRNAexpression of a single miRNA or a profile of two or more miRNAidentified below is established by comparing the profiles of numeroussubjects of the same class (e.g., patients with a certain type and stageof lung cancer or COPD, or a mixture of types and stages) with numeroussubjects of a class from which these individuals must be distinguishedin order to provide a useful diagnosis.

These methods of lung disease screening employ compositions suitable forconducting a simple and cost-effective and non-invasive blood test usingmiRNA expression profiling that could alert the patient and physician toobtain further studies, such as a chest radiograph or CT scan, in muchthe same way that the prostate specific antigen is used to help diagnoseand follow the progress of prostate cancer. The miRNA expression levelsand profiles described herein provide the basis for a variety ofclassifications related to this diagnostic problem. The application ofthese comparative levels and profiles provides overlapping andconfirmatory diagnoses of the type of lung disease, beginning with theinitial test for malignant vs. non-malignant disease.

I. Definitions

“Patient” or “subject” as used herein means a mammalian animal,including a human, a veterinary or farm animal, a domestic animal orpet, and animals normally used for clinical research. More specifically,the subject of these methods and compositions is a human.

“Reference” level, standard or profile as used herein refers to thesource of the reference miRNA. In one embodiment, the reference miRNAstandard is obtained from biological samples selected from a referencehuman subject or population having a non-small cell lung cancer (NSCLC).For example, in one embodiment, the reference standard utilized is astandard or profile derived from the PBMC of a reference human subjector population of human subjects with squamous cell carcinoma or anaverage of multiple subjects with squamous cell carcinoma. In certainembodiments, the reference standard utilized is a standard or profilederived from the PBMC of a reference human subject, or an average ofmultiple subjects, with early stage squamous cell carcinoma. In anotherembodiment, the reference standard is a standard or profile derived fromthe PBMC of a reference human subject, or an average of multiplesubjects, with adenocarcinoma. In another embodiment, the referencestandard is a standard or profile derived from the PBMC of a referencehuman subject, or an average of multiple subjects, with early stageadenocarcinoma.

In another embodiment, the reference miRNA standard is obtained frombiological samples selected from a reference human subject or populationhaving COPD. For example, the reference standard is a standard orprofile derived from the PBMC of a reference human subject, or anaverage of multiple subjects, with COPD. In one embodiment, thereference miRNA standard is obtained from biological samples selectedfrom a reference human subject or population who are healthy and havenever smoked. For example, the reference standard is a standard orprofile derived from the PBMC of a reference human subject, or anaverage of multiple subjects, who are healthy and have never smoked. Inone embodiment, the reference miRNA standard is obtained from biologicalsamples selected from a reference human subject or population who areformer smokers or current smokers with no disease. For example, thereference standard is a standard or profile derived from the PBMC of areference human subject, or an average of multiple subjects, who areformer smokers or current smokers with no disease.

In one embodiment, the reference miRNA standard is obtained frombiological samples selected from a reference human subject or populationhaving benign lung nodules. For example, the reference standard is astandard or profile derived from the PBMC of a reference human subject,or an average of multiple subjects, who have benign lung nodules. In oneembodiment, the reference miRNA standard is obtained from biologicalsamples selected from a reference human subject or population followingsurgical removal of an NSCLC tumor. In one embodiment, the referencemiRNA standard is obtained from biological samples selected from areference human subjects or population prior to surgical removal of anNSCLC tumor. In one embodiment, the reference miRNA standard is obtainedfrom biological samples selected from the same subject who provided atemporally earlier biological sample. In another embodiment, thereference standard is a combination of two or more of the abovereference standards.

The reference standard, in various embodiments, is a mean, an average, anumerical mean or range of numerical means, a numerical pattern, agraphical pattern or an miRNA or mRNA or gene expression profile derivedfrom a reference subject or reference population. Selection of theparticular class of reference standards, reference population, miRNAlevels or profiles depends upon the use to which thediagnostic/monitoring methods and compositions are to be put by thephysician.

“Sample” or “Biological Sample” as used herein means any biologicalfluid or tissue that contains immune cells and/or cancer cells. In oneembodiment, a suitable sample is whole blood. In another embodiment, asuitable sample for use in the methods described herein includesperipheral blood, more specifically peripheral blood mononuclear cells.Other useful biological samples include, without limitation, wholeblood, plasma, serum, saliva, urine, synovial fluid, bone marrow,cerebrospinal fluid, vaginal mucus, cervical mucus, nasal secretions,sputum, semen, amniotic fluid, bronchoalveolar lavage fluid, and othercellular exudates from a subject suspected of having a lung disease.Such samples may further be diluted with saline, buffer or aphysiologically acceptable diluent. Alternatively, such samples areconcentrated by conventional means. It should be understood that the useor reference throughout this specification to any one biological sampleis exemplary only. For example, where in the specification the sample isreferred to as PBMC, it is understood that other samples, e.g., wholeblood, plasma, etc., may also be employed in the same manner.

“Immune cells” as used herein means B-lymphocytes, T-lymphocytes, NKcells, macrophages, mast cells, monocytes and dendritic cells.

As used herein, the term “condition” refers to the absence (healthycondition) or presence of a disease including a lung disease, a lungcancer, the presence of benign nodules or benign tumor growths in thelung, chronic obstructive pulmonary disease (with or without associatedcancer), the existence of a cancerous lung tumor prior to surgery, thepost-surgical condition after removal of a cancerous lung tumor. Wherespecified, any of such conditions can be associated with smoking ornot-smoking.

As used herein, the term “lung disease” refers to a lung cancer orchronic obstructive pulmonary disease, or the presence of lung nodulesor lung lesions due to smoking or some other adverse even in the lungtissue.

As used herein the term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell growth. More specifically, as used herein, the term“cancer” means any lung cancer. In one embodiment, the lung cancer isnon-small cell lung cancer (NSCLC). In a more specific embodiment, thelung cancer type is lung adenocarcinoma (AC). In another embodiment, thelung cancer type is lung squamous cell carcinoma (SCC). In anotherembodiment, the lung cancer is an “early stage” (I or II) NSCLC. Instill another embodiment, the lung cancer is a “late stage” (III or IV)NSCLC. In still another embodiment, the lung cancer is a mixture ofearly and late stages and types of NSCLC.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

By “therapeutic reagent” or “regimen” is meant any type of treatmentemployed in the treatment of cancers with or without solid tumors,including, without limitation, chemotherapeutic pharmaceuticals,biological response modifiers, radiation, diet, vitamin therapy, hormonetherapies, gene therapy, surgical resection, etc.

By “specified miRNAs” as used herein is meant those miRNAs theexpression of which changes (either in an up-regulated or down-regulatedmanner) characteristically in the presence of a condition such as lungdisease. In one embodiment, the specified miRNAs are those reported inTable 1 and FIG. 3. A statistically significant number of suchinformative miRNAs that form a suitable miRNA expression profile for usein the methods and compositions is determined based upon the ability ofsame to discriminate between two or more of the tested referencepopulations.

The term “statistically significant number of miRNAs” in the context ofthis invention differs depending on the degree of change in miRNAexpression observed. The degree of change in miRNA expression varieswith the condition, such as type of lung disease or cancer and with thesize or spread of the cancer or solid tumor. The degree of change alsovaries with the immune response of the individual and is subject tovariation with each individual. The degree of change in expression ofthe specified miRNAs varies with the type of disease diagnosed, e.g.,COPD or NSCLC, and with the size or spread of the cancer or solid tumor.The degree of change also varies with the immune response of theindividual and is subject to variation with each individual. Forexample, in one embodiment of this invention, a change at or greaterthan a 1.2 fold increase or decrease in expression of a miRNA or morethan two such miRNA, or even 3 to about 43 characteristic miRNA, isstatistically significant. In another embodiment, a larger change, e.g.,at or greater than a 1.5 fold, greater than 1.7 fold or greater than 2.0fold increase or decrease in expression of a miRNA or more than two suchmiRNA, or even 3 to about 43 characteristic miRNA, is statisticallysignificant. This is particularly true for cancers without solid tumors.Still alternatively, if a single miRNA is profiled as up-regulated orexpressed significantly in cells which normally do not express themiRNA, such up-regulation of a single miRNA may alone be statisticallysignificant. Conversely, if a single miRNA is profiled as down-regulatedor not expressed significantly in cells which normally do express themiRNA, such down-regulation of a single miRNA may alone be statisticallysignificant.

Thus, the methods and compositions described herein contemplateexamination of the expression level or profile of from 1 to about 50miRNA in a single profile (see Table 1). In one embodiment, asignificant change in the expression level of one of the identifiedmiRNA can be diagnostic of a condition, e.g., lung disease. In anotherembodiment, a significant change in the expression level of two of theidentified miRNAs can indicate a condition, e.g., a lung disease. Inanother embodiment, a significant change in the expression level ofthree of the identified miRNAs can be diagnostic of a lung disease orindicate another condition. In another embodiment, a significant changein the expression level of four or more of the identified miRNAs can bediagnostic of a lung disease or indicate another condition. In anotherembodiment, a significant change in the expression level of at least 5,at least 6, at least 7, at least 8, at least 9 or at least 10 of theidentified miRNAs can be diagnostic of a lung disease or indicateanother condition. In another embodiment, a significant change in theexpression level of about 15 of the identified miRNAs can be diagnosticof a lung disease or indicate another condition. In another embodiment,a significant change in the expression level of about 20 to 40 of theidentified miRNAs can be diagnostic of a lung disease or indicateanother condition. Still other numbers of miRNA changes within thespecifically identified miRNA can be used in diagnosis of lung diseaseor indicate another condition as taught herein.

The term “microarray” refers to an ordered arrangement of hybridizablearray elements. In one embodiment, a microarray comprises polynucleotideprobes that hybridize to the specified miRNA, on a substrate. In anotherembodiment, a microarray comprises multiple primers or antibodies,optionally immobilized on a substrate.

A change in expression of an miRNA required for diagnosis or detectionby the methods described herein refers to an miRNA whose expression isactivated to a higher or lower level in a subject having a condition orsuffering from a disease, specifically lung cancer or NSCLC, relative toits expression in a reference subject or reference standard. miRNAs mayalso be expressed to a higher or lower level at different stages of thesame disease or condition. Expression of specific miRNAs differ betweennormal subjects who never smoked or are current or former smokers, andsubjects suffering from a disease, specifically COPD, benign lungnodules, or cancer, or between various stages of the same disease.Expression of specific miRNAs differ between pre-surgery andpost-surgery patients with lung cancer. Such differences in miRNAexpression include both quantitative, as well as qualitative,differences in the temporal or cellular expression patterns among, forexample, normal and diseased cells, or among cells which have undergonedifferent disease events or disease stages. For the purpose of thisinvention, a significant change in miRNA expression when compared to areference standard is considered to be present when there is astatistically significant (p<0.05) difference in miRNA expressionbetween the subject and reference standard or profile.

In the context of the compositions and methods described herein,reference to “at least two,” “at least five,” etc. of the miRNAs listedin any particular miRNA set means any and all combinations of the miRNAsidentified. Specific miRNAs for the miRNA profile do not have to be inrank order in Table 1 and may be any miRNA identified herein.

One skilled in the art may readily reproduce the compositions andmethods described herein by use of the sequences of the miRNAs, all ofwhich are publicly available from conventional sources, such as MiRBaseor ABI, and are reproduced in Table 1 and in the Sequence Listing.

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment is also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an”, refers to one or more, for example, “anmiRNA,” is understood to represent one or more miRNAs. As such, theterms “a” (or “an”), “one or more,” and “at least one” are usedinterchangeably herein.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts, which provide one skilled in the art witha general guide to many of the terms used in the present application.

II. miRNA Expression and Expression Profiles

The inventors identified diagnostic miRNA biomarkers and expressionprofiles consisting of multiple miRNA biomarkers in the peripheral bloodlymphocytes of lung cancer (NSCLC) and COPD patients, as well as inpopulations of healthy smokers and never smokers, and in pre andpost-surgical patients. The inventors have discovered that various miRNAexpression levels and expression profiles of multiple miRNAs from thePBMCs of lung cancer patients differ significantly from those seen inother populations having non-cancer lung disease or no disease. Changesin the miRNA expression levels or profiles are observed in the normalcirculating PBMC of subjects with early stage solid tumors(adenocarcinoma or squamous cell carcinoma), more readily than can bedetected in circulating tumor cells, which are present in only vanishingsmall numbers in early stage lung cancers.

As detailed in the examples below, the inventors have discovered theidentity of certain miRNA sequences that can serve either as individualbiomarkers to identify an NSCLC from either non-cancerous COPD, or fromotherwise healthy populations of current and former smokers, who mayhave some characteristics of disease due to the smoking history, as wellas from populations of never smokers. It is anticipated that some of themiRNA may serve as biomarkers in diagnostic assays in conjunction withone or more additional miRNA biomarkers, so as to form a characteristicsignature that can distinguish among the various diseased and healthypopulations.

Table 1 identifies the miRNA useful in the methods described herein bymiRBase Accession number, by RNA sequence, by ABI Accession number andby name.

TABLE 1 App. Bio. SEQ ID Sequence miRBase Acc No. Part No. Symbol NOCAUAAAGUAGAAAGCACU MI0000458 / 4373135 miR-142-5p 1 AC MIMAT0000433CUGGCCCUCUCUGCCCUU MI0000804 / 4373049 miR-328 2 CCGU MIMAT0000752UCGGAUCCGUCUGAGCUU MI0000472 / 4373147 miR-127 3 GGCU MIMAT0000446AGCUACAUUGUCUGCUGG MI0000298 / 4373077 miR-221 4 GUUUC MIMAT0000278UCAGUGCAUCACAGAACU MI0000811 / 4373129 miR-148b 5 UUGU MIMAT0000759UCAGUGCACUACAGAACU MI0000253 / 4373130 miR-148a 6 UUGU MIMAT0000243ACUAGACUGAAGCUCCUU MI0000809 / 4373179 miR-151 7 GAGG MIMAT0000757AGCAGCAUUGUACAGGGC MI0000108 / 4373158 miR-103 8 UAUGA MIMAT0007402UCAAGAGCAAUAACGAAA MI0000816 / 4373045 miR-335 9 AAUGU MIMAT0000765UCGUACCGUGAGUAAUAA MI0000471 / 4378064 miR-126 10 UGC MIMAT0000445UAAGGUGCAUCUAGUGCA MI0000072 / 4373118 miR-18a 11 GAUA MIMAT0000072AGAGGUAGUAGGUUGCA MI0000065 / 4373166 let-7d 12 UAGU MIMAT0000065CAAAGAAUUCUCCUUUUG MI0000483 4373112 miR-186 13 GGCUU AAUGACACGAUCACUCCCMI0001448 / 4380926 miR-425-5p 14 GUUGA MIMAT0003393 GCCCCUGGGCCUAUCCUAMI0000812 / 4373046 miR-331 15 GAA MIMAT0000760 CAAAGUGCUUACAGUGCA4373119 miR-17-5p 16 GGUAGU UAGCAGCACAUCAUGGUU MI0000438 / 4373122miR-15b 17 UACA MIMAT0000417 UGAGGUAGUAGGUUGUA MIMAT0000062 4373169 let-7a 18 UAGUU AAACCGUUACCAUUACUG MI0001729 4373209 miR-451 19 AGUUUGGCAAGAUGCUGGCAUAG MI0000089 4373190 miR-31 20 CUG UCCUGUACUGAGCUGCCCMI0002470 4378096 miR-486 21 CGAG CAAAGUGCUCAUAGUGCA MI0001519 / 4373263miR-20b 22 GGUAG MIMAT0001413 CUUUCAGUCGGAUGUUUA MIMAT0000693 4373057miR-30e-3p 23 CAGC AGCUCGGUCUGAGGCCCC MI0001445 / 4373015 miR-423 24UCAG MIMAT0001340 UACAGUAGUCUGCACAUU 4378068 miR-199a 25 GGUUAAAGUGCUGUUCGUGCAG MI0000095 4373012 miR-93 26 GUAG AAGGAGCUCACAGUCUAUMI0000086 / 4373067 miR-28 27 UGAG MIMAT0000085 UAGCUUAUCAGACUGAUGMI0000077 / 4373090 miR-21 28 UUGA MIMAT0000076 AGCUACAUCUGGCUACUGMI0000299 4373076 miR-222 29 GGUCUC UGUAAACAUCCUUGACUG MIMAT00006924373058 miR-30e-5p 30 GA CCCAUCUGGGGUGGCCUG 4380958 miR-594 31 UGACUUUUCCGUCUCAGUUACUUUA MI0000802 4373041 miR-340 32 UAGCC CAUUAUUACUUUUGGUACMI0000471 4373269 miR-126 33 GCG CAGUGCAAUGUUAAAAGG MI0000448 / 4373145miR-130a 34 GCAU MIMAT0000425 CAUUGCACUUGUCUCGGU MI0000082 / 4373071miR-25 35 CUGA MIMAT0000081 UACCCAUUGCAUAUCGGA MI0003684 / 4380925miR-660 36 GUUG MIMAT0003338 AACAUUCAUUGUUGUCGG MI0003139/ 4373180miR-181d 37 UGGGUU MIMAT0002821 UCCCUGAGACCCUUUAAC MI0000469 / 4373149miR-125a 38 CUGUG MIMAT0000443 UUCACAGUGGCUAAGUUC MI0000440 / 4373068miR-27b 39 UGC MIMAT0000419 CAUGCCUUGAGUGUAGGA MI0003205 / 4380928miR-532 40 CCGU MIMAT0002888 UGGCUCAGUUCAGCAGGA MIMAT0000080 4373072miR-24 41 ACAG AACAUUCAUUGCUGUCGG MIMAT0000257 4373116 miR-181b 42 UGGGAAGCUGCCAGUUGAAGAA MI0000078 / 4373079 miR-22 43 CUGU MIMAT0000077UGAGGUAGUAGGUUGUA MIMAT0000064 miR-let-7c 44 UGGUU UGGCAGUGUCUUAGCUGGMIMAT0000255 miR-34a 45 UUGU UUCCUAUGCAUAUACUUC MIMAT0002810 miR-202* 46UUUG UGAGACCUCUGGGUUCUG MIMAT0003886 miR-769-5p 47 AGCUGUCCCUCUCCAAAUGUGU MIMAT0003312 miR-642 48 CUUG

Table 1 and FIG. 3 provide a list of identified miRNA biomarkers, andidentify them by their conventional miRNA names, as well as by theirAccession Nos. in the miRBase and ABI databases. The expression of eachmiRNA for each of five subject populations is provided using anarbitrary expression number obtained from hierarchical clustering data.In one embodiment of the diagnostic methods described herein, at least a1.2 fold difference between the miRNA expression levels of twopopulations is considered significant for purposes of distinguishingbetween the populations. In another embodiment of the diagnostic methodsdescribed herein, at least a 1.5 fold difference between the miRNAexpression levels of two populations is considered significant forpurposes of distinguishing between the populations. In anotherembodiment of the diagnostic methods described herein, at least a 1.7fold difference between the miRNA expression levels of two populationsis considered significant for purposes of distinguishing between thepopulations. In still another embodiment of the diagnostic methodsdescribed herein, a >2 fold difference between the miRNA expressionlevels of two populations is considered significant for purposes ofdistinguishing between the populations.

In one embodiment, the following miRNA biomarkers are particularlyuseful alone or in combinations forming a profile for distinguishingbetween subjects with COPD and subjects with any NSCLC: miR-142-5p,miR-328, miR-127, miR-221, miR-148b, miR-148a, miR-151, miR-103,miR-18a, miR-let-7d, miR-186, miR-15b, miR-let-7a, miR-451, miR-20b,miR-30e-3p, and miR-27b.

In another embodiment, the following miRNA biomarkers are particularlyuseful alone or in combination forming a profile for distinguishingbetween subjects with adenocarcinoma vs. squamous cell carcinoma:miR-142-5p, miR-328, miR-148b, miR-151, miR-335, miR-221, miR-126,miR-425-5p, miR-331, miR-31, miR-486, miR-199a and miR-222.

In still another embodiment certain miRNA biomarkers are particularlyuseful alone or in combinations forming a profile for distinguishingbetween subjects with COPD and SCC, such as miR-142-5p, miR-328,miR-127, miR-221, miR-148b, miR-148a, miR-151, miR-103, miR-335,miR-126, miR-18a, miR-let-7d, miR-186, miR-425-5p, miR-331, miR-17-5p,as well as others characterized as max ratio COPD/SCC in FIG. 3.

In another embodiment, miR-142-5p and miR-486 are useful biomarkers fordistinguishing between healthy smokers and subjects with adenocarcinoma.

In another embodiment, miR-328, miR-221, miR-148b, miR-151, miR-let-7dand miR181d are useful biomarkers for distinguishing between healthysmokers and subjects with squamous cell carcinoma.

In another embodiment, one or more of the miRNAs: hsa-miR-let-7c,hsa-miR-34a, hsa-miR-202, hsa-miR-769-5p, and hsa-miR-642 are useful indistinguishing between lung cancer patients pre-surgery and patients whohave had surgery to remove the lung tumors. These biomarkers are usefulto monitor the post-surgery progress of this class of subjects.

Similar biomarkers may be selected by calculation of the ratios ofexpression between selected populations from the arbitrary unitsprovided in FIG. 3 so as to identify expression of miRNA in this groupsthat are particularly useful alone, or in combinations in profiles todistinguish between, e.g., subjects who are relatively healthy but mayhave benign lesions due to smoking (S) and subjects with NSCLC ingeneral, or a particularly cancer, such as AC or SCC. Still other miRNAbiomarkers are useful alone or in a profile with other biomarkers todistinguish between subjects with lung disease, e.g., COPD but notcancer NSCLC, etc.

It should be understood that based upon the teachings herein the miRNAexpression signatures or profiles identified herein for use inidentifying any one of the conditions discussed, e.g., an NSCLC or COPD,may be further adjusted to reduce the numbers of miRNA sequencesnecessary to increase accuracy of diagnosis. Such diagnostic methods andprofiles may also be used in conjunction with other known methods ofdiagnosing or detecting NSCLC and COPD, or any other condition discussedherein to further increase accuracy of diagnosis.

III. Methods of Detecting/Quantifying miRNA

Methods that may be employed in obtaining, detecting and quantifyingmiRNA expression are known and may be used to accomplish the diagnosticgoals of the present invention. See, for example, the techniquesdescribed in the examples below, as well as in e.g., WO2008/073923; USPublished Patent Application No. 2006/0134639, and U.S. Pat. No.6,040,138, among others.

For example, the biological samples may be collected using theproprietary PaxGene Blood RNA System (PreAnalytiX, a Qiagen, BDcompany). The PAXgene Blood RNA System comprises two integratedcomponents: PAXgene Blood RNA Tube and the PAXgene Blood RNA Kit. Bloodsamples are drawn directly into PAXgene Blood RNA Tubes via standardphlebotomy technique. These tubes contain a proprietary reagent thatimmediately stabilizes intracellular RNA, minimizing the ex-vivodegradation or up-regulation of RNA transcripts. The ability toeliminate freezing, batch samples, and to minimize the urgency toprocess samples following collection, greatly enhances lab efficiencyand reduces costs.

Thereafter, the miRNA are detected and/or measured using a variety ofassays. The most sensitive and most flexible quantitative method isreal-time polymerase chain reaction (RT-PCR), which can be used tocompare miRNA levels in different sample populations, in normal andtumor tissues, with or without drug treatment, to characterize patternsof miRNA expression, to discriminate between closely related miRNAs, andto analyze RNA structure. This method can be employed by usingconventional RT-PCR assay kits according to manufacturers' instructions,such as TaqMan® RT-PCR (Applied Biosystems).

The first step is the isolation of RNA from a target sample (e.g.,typically total RNA isolated from human PBMC in this case). RNA can beextracted, for example, from frozen or archived paraffin-embedded andfixed (e.g., formalin-fixed) tissue samples. General methods for mRNAextraction are well known in the art, e.g., in standard textbooks ofmolecular biology, including methods for RNA extraction from paraffinembedded tissues. In particular, RNA isolation can be performed using apurification kit, buffer set and protease from commercial manufacturers,according to the manufacturer's instructions. Exemplary commercialproducts include TRI-REAGENT, Siegen RNeasy mini-columns, MASTERPUREComplete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.),Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNA Stat-60(Tel-Test). Conventional techniques such as cesium chloride densitygradient centrifugation may also be employed.

Next, in the reverse transcription step, cDNA is reverse transcribedfrom mRNA samples using primers specific for the miRNAs to be detected.Methods for reverse transcription are well known in the art, e.g., instandard textbooks of molecular biology. Briefly, RNA is first incubatedwith a primer at 70° C. to denature RNA secondary structure and thenquickly chilled on ice to let the primer anneal to the RNA. Othercomponents are added to the reaction including dNTPs, RNase inhibitor,reverse transcriptase and reverse transcription buffer. The reversetranscription reaction is extended at 42° C. for 1 hr. The reaction isthen heated at 70° C. to inactivate the enzyme. Optionally, the templateRNA may be removed by treating the reverse transcription reaction withRNase H before using the reaction in the real time PCR reaction.

In the RT-PCR step, PCR products are amplified from the cDNA samples.PCR product accumulation is measured through a dual-labeled fluorigenicprobe (i.e., TAQMAN® probe). Real time PCR is compatible both withquantitative competitive PCR, where an internal competitor for eachtarget sequence is used for normalization, and with quantitativecomparative PCR using a normalization miRNA contained within the sample,or a housekeeping miRNA for RT-PCR. For further details see, e.g. Heldet al., Genome Research 6:986 994 (1996).

TaqMan® RT-PCR can be performed using commercially available equipment.In a preferred embodiment, the 5′ nuclease procedure is run on areal-time quantitative RCR device such as the ABI PRISM 7900® SequenceDetection System®. The system amplifies samples in a 96 (or 384)-wellformat on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optic cablesfor all 96 (or 384) wells, and detected at the CCD. The system includessoftware for running the instrument and for analyzing the data.5′-Nuclease assay data are initially expressed as Ct, or the thresholdcycle. As discussed above, fluorescence values are recorded during everycycle and represent the amount of product amplified to that point in theamplification reaction. The point when fluorescent signal is firstrecorded as statistically significant is the threshold cycle (Ct).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed as a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of miRNA expression are mRNAs for the housekeepingmiRNAs glyceraldehydes-3phosphate-dehydrogenase (GAPDH) and β-actin.

The steps of a representative protocol from profiling miRNA expressionusing fixed, paraffin-embedded tissues as the RNA source, including mRNAisolation, purification, primer extension and amplification are known tothose of skill in the art. Briefly, a representative process starts withcutting about 10 μm thick sections of paraffin-embedded tumor tissuesamples. The RNA is then extracted, and protein and DNA are removed.After analysis of the RNA concentration, RNA repair and/or amplificationsteps may be included, if necessary, and RNA is reverse transcribedusing miRNA specific promoters followed by RT-PCR.

The specific techniques identified in Example 1 below demonstrate thestate of the art. However, other conventional methods of miRNAisolation, detection and quantification can be employed in thesemethods.

Still other methods of detecting and/or measuring miRNA may be employed,using antibodies or fragments thereof. As used herein, the term“antibody” refers to an intact immunoglobulin having two light and twoheavy chains or any fragments thereof. Thus a single isolated antibodyor fragment may be a polyclonal antibody, a high affinity polyclonalantibody, a monoclonal antibody, a synthetic antibody, a recombinantantibody, a chimeric antibody, a humanized antibody, or a humanantibody. The term “antibody fragment” refers to less than an intactantibody structure, including, without limitation, an isolated singleantibody chain, a single chain Fv construct, a Fab construct, a lightchain variable or complementarity determining region (CDR) sequence,etc. A recombinant molecule bearing a sequence that binds to the miRNAmay also be used in these methods. It should be understood that anyantibody, antibody fragment, or mixture thereof that binds a specifiedmiRNA as defined herein may be employed in the methods of the presentinvention, regardless of how the antibody or mixture of antibodies wasgenerated.

Similarly, methods using genomic or other hybridization probes toidentify the miRNA sequences are useful herein. In another embodiment, asuitable assay detection assay is an immunohistochemical assay, ahybridization assay, a counter immuno-electrophoresis, aradioimmunoassay, radioimmunoprecipitation assay, a dot blot assay, aninhibition of competition assay, or a sandwich assay.

Any of the methods described above or otherwise herein may be performedby a computer processor or computer-programmed instrument that generatesnumerical or graphical data useful in the diagnosis or detection of thecondition or differentiation between two conditions.

IV. Compositions of the Invention

The methods for diagnosing or detecting lung disease utilizing definedmiRNA biomarker expression levels or profiles of multiple miRNAbiomarkers permits the development of simplified diagnostic tools fordiagnosing or detecting lung cancer, e.g., NSCLC or diagnosing ordetecting a specific stage (early, stage I, stage II or late stage) oflung cancer, diagnosing or detecting a specific type of lung cancer(e.g., AC vs. SCC), distinguishing between COPD and lung cancer orbenign lung nodules and lung cancer, and/or monitoring the effect oftherapeutic or surgical intervention for determination of furthertreatment or evaluation of the likelihood of recurrence of the cancer.

In one aspect, diagnostic reagent is capable of specifically complexingwith or identifying an miRNA of Table 1. In another embodiment, thereagent comprises a ligand capable of complexing with, hybridizing to,or identifying an miRNA of Table 1. In another embodiment, the miRNAinclude hsa-miR-148a, hsa-miR-142-5p, hsa-miR-221, hsa-miR-let-7d,hsa-miR-let-7a, hsa-miR-328, hsa-miR-let-7c, hsa-miR-34a, hsa-miR-202,hsa-miR-769-5p, hsa-miR-642, and a combination of two or more thereof.The reagent, in one embodiment, is an amplification nucleic acid primer(such as an RNA primer) or primer pair that amplifies and detects anucleic acid sequence of said miRNA. In another embodiment, the reagentis a polynucleotide probe that hybridizes to the miRNA nucleic acidsequence. In another embodiment, the reagent is an antibody or fragmentof an antibody. The reagent can include multiple said primers, probes orantibodies, each specific for at least one miRNA of Table 1. In certainembodiments, the reagent is immobilized on a substrate. Exemplarysubstrates include a microarray, chip, microfluidics card, or chamber.

Optionally, the diagnostic reagent can be associated with a conventionaldetectable label. As used herein, “labels” or “reporter molecules” arechemical or biochemical moieties useful for labeling a nucleic acid(including a single nucleotide), polynucleotide, oligonucleotide, orprotein ligand, e g, amino acid or antibody. “Labels” and “reportermolecules” include fluorescent agents, chemiluminescent agents,chromogenic agents, quenching agents, radionucleotides, enzymes,substrates, cofactors, inhibitors, magnetic particles, and othermoieties known in the art. “Labels” or “reporter molecules” are capableof generating a measurable signal and may be covalently or noncovalentlyjoined to an oligonucleotide or nucleotide (e g, a non-naturalnucleotide) or ligand.

A kit or microarray useful in the methods described herein can includeat least two such diagnostic reagents, each reagent specific for adifferent miRNA of Table 1 or FIG. 3. In still another embodiment, a kitor microarray includes at least 3, at least 4, at least 5, at least 6,at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, at least 20, atleast 25 or more such diagnostic reagents, each reagent specific for adifferent miRNA. One of skill in the art will recognize that allintegers occurring between the numbers specified above are included inthis disclosure, even if not specifically recited herein.

In another aspect, a composition for diagnosing or detecting lung cancerin a mammalian subject includes at least two PCR primers or probes. Eachprimer or probe amplifies a different polynucleotide sequence from amiRNA expression product of at least two miRNAs from TABLE 1 found inthe peripheral blood mononuclear cells (PBMC) of the subject. ThesemiRNAs are selected to form a miRNA expression profile or signaturewhich is distinguishable between a subject having lung cancer and aselected reference population or standard. Changes in expression in theindividual miRNAs or miRNA expression profile of a tested subject fromthat of a reference miRNA expression profile are correlated with a lungdisease, such as non-small cell lung cancer (NSCLC).

In one embodiment of this composition, the primers are those that targetmiRNAs selected from among the miRNAs identified in TABLE 1. Thiscollection of miRNAs includes those for which expression is altered(i.e., increased or decreased) versus the same miRNA biomarkerexpression in the PBMC of a reference. In one embodiment, PCR primersand probes are provided to detect at least two miRNAs from TABLE 1 foruse in the composition. In another embodiment, PCR primers and probesare provided to detect at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 miRNAs from TABLE 1 for use in thecomposition. In still another embodiment, PCR primers and probes areprovided more than 20, but less than 43 miRNAs from TABLE 1 for use inthe composition. The primers or probes used to target the selectedmiRNAs from the TABLE 1 and FIG. 3 need not be in rank order; rather anycombination that clearly targets miRNAs that show a difference inexpression between the reference population to the diseased populationis useful in such a composition.

In one embodiment, the composition contains primers or probes useful toidentify at least two of the following miRNAs: hsa-miR-148a,hsa-miR-142-5p, hsa-miR-221, hsa-miR-let-7d, hsa-miR-let-7a andhsa-miR-328. In another exemplary embodiment, the composition containsprimers useful to identify at least two or more of the following miRNAs:miR-142-5p, miR-328, miR-127, miR-221, miR-148b, miR-148a, miR-151,miR-103, miR-18a, miR-let-7d, miR-186, miR-15b, miR-let-7a, miR-451,miR-20b, miR-30e-3p, and miR-27b. In another exemplary embodiment, thecomposition contains primers useful to identify at least two or more ofthe following miRNAs: miR-142-5p, miR-328, miR-148b, miR-151, miR-335,miR-221, miR-126, miR-425-5p, miR-331, miR-31, miR-486, miR-199a andmiR-222. In another exemplary embodiment, the composition containsprimers useful to identify at least two or more of the following miRNAs:miR-142-5p, miR-328, miR-127, miR-221, miR-148b, miR-148a, miR-151,miR-103, miR-335, miR-126, miR-18a, miR-let-7d, miR-186, miR-425-5p,miR-331, miR-17-5p. In another exemplary embodiment, the compositioncontains primers useful to identify at least two or more of thefollowing miRNAs: miR-142-5p and miR-486. In another exemplaryembodiment, the composition contains primers useful to identify at leasttwo or more of the following miRNAs: miR-328, miR-221, miR-148b,miR-151, miR-let-7d and miR181d. In another embodiment, the compositioncontains primers used to identify two or more of hsa-miR-let-7a,hsa-miR-328, hsa-miR-let-7c, hsa-miR-34a, hsa-miR-202, hsa-miR-769-5p,hsa-miR-642. As discussed above, other specific compositions containingvarious combinations of the miRNA disclosed in TABLE 1 are encompassed.

As provided above, the reference may be any population class asdescribed above in “Definitions”.

The composition, which can be presented in the format of a microfluidicscard, a chip or chamber preferably employs the RT-PCR techniquesdescribed above. In one aspect, such a format is a diagnostic assayusing TAQMAN® Quantitative PCR low density arrays. Preliminary resultssuggest the number of miRNAs required is compatible with theseplatforms. When a sample of PBMC from a selected patent subject iscontacted with the primers and probes in the composition, PCRamplification of targeted miRNAs in the miRNA expression profile fromthe patient occurs. The composition thus permits detection of changes inexpression in the miRNAs in the miRNA expression profile from that of areference miRNA expression standard or profile. Significant changes inthe miRNA expression of the one or more miRNA biomarkers in thepatient's PBMC from that of the reference correlate with a diagnosis ofnon-small cell lung cancer (NSCLC), a stage of such cancer, a type ofsuch cancer, or a non-cancerous condition, such as COPD, benign lunglesions or nodules, or none of these conditions.

For use in the above-noted compositions the PCR primers and probes arepreferably designed based upon the miRNA sequences or theircomplement(s) identified in TABLE 1. The design of the primer and probesequences is within the skill of the art once the particular miRNAtarget is selected. The particular methods selected for the primer andprobe design and the particular primer and probe sequences are notlimiting features of these compositions. A ready explanation of primerand probe design techniques available to those of skill in the art byresort to general texts as well as commercial manufacturers (e.g.,Applied Biosystems).

In one embodiment, optimal PCR primers and probes used in thecompositions described herein are about 12-22 bases in length, andcontain about 20-80%, such as, for example, about 50-60% G+C bases.Melting temperatures of between 50 and 80° C., e.g. about 50 to 70° C.are typically preferred.

These compositions may be used to diagnose NSCLC lung cancer of stage Ior stage II NSCLC. Further these compositions are useful to provide asupplemental or original diagnosis in a subject having lung nodules ofunknown etiology.

V. Diagnostic Methods

All of the above-described compositions provide a variety of diagnostictools which permit a blood-based, non-invasive assessment of diseasestatus in a subject. Use of these compositions in diagnostic tests,which may be coupled with other screening tests, such as a chest X-rayor CT scan, increase diagnostic accuracy and/or direct additionaltesting. In other aspects, the diagnostic compositions and toolsdescribed herein permit the prognosis of disease, monitoring response tospecific therapies, and regular assessment of the risk of recurrence.The methods and use of the compositions described herein also permit theevaluation of changes in diagnostic miRNA levels or profilespre-therapy, pre-surgery and/or at various periods during therapy andpost therapy samples and identifies a miRNA expression profile orsignature that may be used to assess the probability of recurrence.

In one embodiment, a method of diagnosing or detecting or assessing acondition in a mammalian subject comprises detecting in a biologicalsample of the subject, or from an miRNA expression profile generatedfrom the sample, the expression level of an miRNA nucleic acid sequenceidentified in Table 1; and comparing the miRNA expression level or miRNAexpression profile in the subject's sample to a reference miRNAstandard. A change in expression of the subject's sample miRNA from thatin the reference miRNA standard indicates a diagnosis or prognosis of acondition mentioned above. In certain embodiments, the condition is alung cancer, chronic obstructive pulmonary disease (COPD), or benignlung nodules. These methods may be employed using the biological samplesdiscussed above. In certain embodiments, the biological sample is wholeblood, peripheral blood mononuclear cells, plasma and serum.

As discussed above, this method involves in certain embodiments,measuring the expression level of one or more specified miRNA in thesubject's sample. In other embodiments, the detecting, measuring orcomparing steps of the method are repeated multiple times. For example,in certain embodiments, the miRNA levels are detected or measured in aseries of samples of said subject taken at different times. This permitsidentification of a pattern of altered expression of said miRNA from aselected reference miRNA standard.

In still other embodiments, the detecting or measuring step involvescontacting a biological sample from the subject with a diagnosticreagent, such as those described above that identifies or measures themiRNA expression level in the sample. In certain embodiments, thecontacting step involves or comprises forming a direct or indirectcomplex in said biological samples between a diagnostic reagent for saidmiRNA and the miRNA in the sample. Thereafter, the method measures alevel of the complex in a suitable assay, such as described herein.

In one embodiment, the method involves diagnosing or detecting the oneor more, or a pattern made up of one or more, of the miRNA nucleic acidsequences: hsa-miR-148a, hsa-miR-142-5p, hsa-miR-221, hsa-miR-let-7d,hsa-miR-let-7a, hsa-miR-328, hsa-miR-let-7c, hsa-miR-34a, hsa-miR-202,hsa-miR-769-5p, hsa-miR-642, and a combination of two or more thereof.

In certain embodiments of these methods, the miRNA(s) is differentiallyexpressed in two or more of the conditions selected from no lung diseasewith no history of smoking, no lung disease with a history of smoking,lung cancer, chronic obstructive pulmonary disease (COPD), benign lungnodules, lung cancer prior to tumor resection, and lung cancer followingtumor resection.

Depending on the conditions being assessed by the methods, the referencemiRNA standard is obtained from a reference subject or referencepopulation such as (a) a reference human subject or population having anon-small cell lung cancer (NSCLC); (b) a reference human subject orpopulation having COPD, (c) a reference human subject or population whoare healthy and have never smoked, (d) a reference human subject orpopulation who are former smokers or current smokers with no disease;(e) a reference human subject or population having benign lung nodules;(f) a reference human subject or population following surgical removalof an NSCLC tumor; (g) a reference human subjects or population prior tosurgical removal of an NSCLC tumor; and (h) the same subject whoprovided a temporally earlier biological sample.

Thus, in one aspect, a method is provided for diagnosing or detecting alung disease in a mammalian subject comprising detecting in a humansubject's sample, e.g., peripheral blood mononuclear cells (PBMC) orwhole blood, or from an miRNA expression profile generated from thesample, the expression level of at least one of the miRNA nucleic acidsequences identified in Table 1 or FIG. 3; comparing the miRNAexpression level or profile in the sample to an average expression levelor standard of the same miRNA in a reference sample or profile. A changein expression (e.g., increased expression or decreased expression,depending upon the disease and miRNA biomarker involved) of at least onethe miRNA from that in the reference is indicative of a non small celllung cancer (NSCLC) or chronic obstructive pulmonary disease (COPD).This method may also be employed to identify a type or stage of NSCLC,as well as enable diagnosis of the absence of malignancy. This methodmay be performed using the appropriately selected compositions describedabove.

In another embodiment, a method is provided for diagnosing or detectinga lung disease in a mammalian subject comprising detecting in a sampleof the subject's peripheral blood mononuclear cells (PBMC) or wholeblood, or from an miRNA expression profile generated from the sample,the expression level of at least one of the miRNA nucleic acid sequencesselected from the group consisting of hsa-miR-148a, hsa-miR-142-5p,hsa-miR-221, hsa-miR-let-7d, hsa-miR-let-7a and hsa-miR-328; andcomparing the miRNA expression level in the sample to an averageexpression level or standard of the same miRNA in a reference sample orprofile. A change in expression of at least one the miRNA from that inthe reference is indicative of a non small cell lung cancer (NSCLC) orchronic obstructive pulmonary disease (COPD).

In other embodiments of these methods, the assessment or identificationof miRNAs to distinguish or identify a condition may be coupled with theassessment or identification of mRNA sequences or gene sequences or anexpression profile of same characteristic of the same condition. Forexample, the methods described above may further comprise additionalsteps. For example, the method can employ detecting in the biologicalsample of the subject, or from a gene expression profile generated fromthe sample, the expression level of a gene associated with thecondition. The gene expression level or gene profile of the subject'ssample is then compared to a reference gene standard; and the expressionlevel or profile of the miRNA obtained as above is correlated with theexpression level or profile of the gene or mRNA sequences. In such amethod, the combined changes in expression of the miRNA and the genefrom their levels of expression in the reference miRNA standard andreference gene standard, respectively, indicates a diagnosis orprognosis of the condition. See e.g., International patent publicationNo. Pub No. WO 2009/075799, among other gene sequences that may becorrelated with the miRNAs of these methods to enhance or make adiagnosis or detect a specific condition.

More specific embodiments of these diagnostic methods are describedbelow.

A. Methods of Diagnosing or Detecting NSCLC

Thus, in one embodiment, a method of diagnosing or detecting squamouscell carcinoma in a mammalian subject involves detecting in a sample ofthe subject's peripheral blood mononuclear cells (PBMC), or from anmiRNA expression profile generated from the sample, the expression levelof the miRNA nucleic acid sequences hsa-miR-148a. In one embodiment, amethod of diagnosing or detecting squamous cell carcinoma in a mammaliansubject involves detecting in a sample of the subject's whole blood, orfrom an miRNA expression profile generated from the sample, theexpression level of the miRNA nucleic acid sequences hsa-miR-148a. ThemiRNA expression level of the specified miRNA in the sample is comparedto an average expression level of the same miRNA in a reference sampleor profile. Where the reference standard or profile is from one or moresubjects with chronic obstructive pulmonary disease (COPD), a decreasein expression of the miRNA from that in the reference is indicative ofsquamous cell carcinoma. Where the reference standard or profile is fromone or more healthy subjects who have never smoked, a decrease inexpression of the miRNA from that in the reference is indicative ofsquamous cell carcinoma. Where the reference standard or profile is fromone or more former or current smokers who have no disease, a decrease inexpression of the miRNA from that in the reference is indicative ofsquamous cell carcinoma. Where the reference standard is a standard orprofile derived from the PBMC of a reference human subject, or anaverage of multiple subjects, who have benign lung nodules, a decreasein expression of the miRNA from that in the reference is indicative ofsquamous cell carcinoma. Where the reference standard or profile is fromone or more subjects with early stage adenocarcinoma, a decrease inexpression of the miRNA from that in the reference is indicative ofsquamous cell carcinoma. In monitoring the progress of an SCC patientundergoing treatment, the expression levels of the miRNA may be comparedin earlier and later biological samples from the subject. An increase inexpression of this miRNA in the later samples may indicate that thetherapeutic regimen is effective. The opposite is true with successivedecreases in expression.

Thus in the diagnosis of NSCLC from other non-cancerous lung diseases,this miRNA biomarker alone or in combination with other miRNA biomarkersin a profile is useful in diagnostic methods.

In still another aspect, a method of diagnosing or detecting non-smallcell lung cancer (NSCLC) in a mammalian subject involves detecting in asample of the subject's peripheral blood mononuclear cells (PBMC), orfrom an miRNA expression profile generated from the sample, theexpression level of the miRNA nucleic acid sequences hsa-miR-let-7a. Inanother aspect, a method of diagnosing or detecting non-small cell lungcancer (NSCLC) in a mammalian subject involves detecting in a sample ofthe subject's whole blood, or from an miRNA expression profile generatedfrom the sample, the expression level of the miRNA nucleic acidsequences hsa-miR-let-7a. The miRNA expression level of the specifiedmiRNA in the sample is compared to an average expression level of thesame miRNA in a reference sample or profile. The miRNA expression levelof the specified miRNA in the sample is compared to an averageexpression level of the same miRNA in a reference standard or profile.Where the reference standard or profile is from one or more subjectswith chronic obstructive pulmonary disease (COPD), a decrease inexpression of the miRNA from that in the reference is indicative ofNSCLC. Where the reference standard is a standard or profile derivedfrom the PBMC of a reference human subject, or an average of multiplesubjects, who have benign lung nodules, a decrease in expression of themiRNA from that in the reference is indicative of NSCLC. Where thereference standard or profile is from one or more healthy subjects whohave never smoked, a decrease in expression of the miRNA from that inthe reference is indicative of NSCLC. Where the reference standard orprofile is from one or more former or current smokers who have nodisease, a decrease in expression of the miRNA from that in thereference indicates a diagnosis of NSCLC, e.g., squamous cell carcinomaor adenocarcinoma. This biomarker may similarly be employed in assessingtherapeutic efficacy in multiple samples from the treated subject asdescribed above.

Thus in the diagnosis of NSCLC from other non-cancerous lung diseases,this miRNA biomarker alone or in combination with other miRNA biomarkerssuch as miR-148a in a profile is useful in diagnostic methods.

In yet a further aspect, a method of diagnosing or detecting squamouscell carcinoma in a mammalian subject is provided, comprising detectingin a subject's sample, e.g., whole blood or peripheral blood mononuclearcells (PBMC), or from an miRNA expression profile generated from thesample, the expression level of the miRNA nucleic acid sequenceshsa-miR-let-7d. The miRNA expression level of the specified miRNA in thesample is compared to an average expression level of the same miRNA in areference sample or profile. Where the reference standard or profile isfrom one or more subjects with chronic obstructive pulmonary disease(COPD), a decrease in expression of the miRNA from that in the referenceis indicative of SCC. Where the reference standard is a standard orprofile derived from the PBMC of a reference human subject, or anaverage of multiple subjects, who have benign lung nodules, a decreasein expression of the miRNA from that in the reference is indicative ofSCC. Where the reference standard or profile is from one or more healthysubjects who have never smoked, a decrease in expression of the miRNAfrom that in the reference is indicative of SCC. Where the referencestandard or profile is from one or more former or current smokers whohave no disease, a decrease in expression of the miRNA from that in thereference indicates a diagnosis of SCC. This biomarker may similarly beemployed in assessing therapeutic efficacy in multiple samples from thetreated subject as described above.

In another aspect, a method of diagnosing or detecting squamous cellcarcinoma in a mammalian subject is provided, comprising detecting in asubject's sample, e.g., whole blood or peripheral blood mononuclearcells (PBMC), or from an miRNA expression profile generated from thesample, the expression level of the miRNA nucleic acid sequenceshsa-miR-221. The miRNA expression level of the specified miRNA in thesample is compared to an average expression level of the same miRNA in areference sample or profile. Where the reference standard or profile isfrom one or more subjects with chronic obstructive pulmonary disease(COPD), a decrease in expression of the miRNA from that in the referenceis indicative of SCC. Where the reference standard is a standard orprofile derived from the PBMC of a reference human subject, or anaverage of multiple subjects, who have benign lung nodules, a decreasein expression of the miRNA from that in the reference is indicative ofSCC. Where the reference standard or profile is from one or more healthysubjects who have never smoked, a decrease in expression of the miRNAfrom that in the reference is indicative of SCC. Where the referencestandard or profile is from one or more former or current smokers whohave no disease, a decrease in expression of the miRNA from that in thereference indicates a diagnosis of SCC. This biomarker may similarly beemployed in assessing therapeutic efficacy in multiple samples from thetreated subjects as described above.

Thus in the diagnosis of SCC from other non-cancerous lung diseases, oneor both the miRNA biomarkers miR-let-7d or miR-221 alone or incombination with other miRNA biomarkers in a profile is useful indiagnostic methods.

In another aspect, a method of diagnosing or detecting adenocarcinoma ina mammalian subject is provided, comprising detecting in a biologicalsample of the subject, e.g., whole blood or peripheral blood mononuclearcells (PBMC), or from an miRNA expression profile generated from thesample, the expression level of the miRNA nucleic acid sequenceshsa-miR-142-5p. The miRNA expression level of the specified miRNA in thesample is compared to an average expression level of the same miRNA in areference sample or profile. Where the reference standard or profile isfrom one or more subjects with chronic obstructive pulmonary disease(COPD), a decrease in expression of the miRNA from that in the referenceis indicative of AC. Where the reference standard is a standard orprofile derived from the PBMC of a reference human subject, or anaverage of multiple subjects, who have benign lung nodules, a decreasein expression of the miRNA from that in the reference is indicative ofAC. Where the reference standard or profile is from one or more healthysubjects who have never smoked, a decrease in expression of the miRNAfrom that in the reference is indicative of AC. Where the referencestandard or profile is from one or more former or current smokers whohave no disease, a decrease in expression of the miRNA from that in thereference indicates a diagnosis of AC. This biomarker may similarly beemployed in assessing therapeutic efficacy in multiple samples from thetreated subject as described above.

Thus in the diagnosis of AC from other non-cancerous lung diseases, themiRNA biomarker miR-142-5p alone or in combination with other miRNAbiomarkers in a profile is useful in diagnostic methods.

In another aspect, a method of diagnosing or detecting the type of NSCLCin a mammalian subject is provided, comprising detecting in a sample ofthe subject's peripheral blood mononuclear cells (PBMC), or from anmiRNA expression profile generated from the sample, the expression levelof the miRNA nucleic acid sequences hsa-miR-148a. The miRNA expressionlevel of the specified miRNA in the sample is compared to an averageexpression level of the same miRNA in a reference sample or profile.Where the reference standard or profile is from one or more subjectsdiagnosed with SCC, a decrease in expression of the miRNA from that inthe reference is indicative of a diagnosis of adenocarcinoma. Where thereference standard or profile is from one or more subjects diagnosedwith AC, an increase in expression of the miRNA from that in thereference is indicative of a diagnosis of SCC. This biomarker maysimilarly be employed in assessing therapeutic efficacy in multiplesamples from the treated subject as described above.

In another aspect, a method of diagnosing or detecting adenocarcinoma ina mammalian subject is provided, comprising detecting in a biologicalsample of the subject, e.g., whole blood or peripheral blood mononuclearcells (PBMC), or from an miRNA expression profile generated from thesample, the expression level of the miRNA nucleic acid sequenceshsa-miR-328. The miRNA expression level of the specified miRNA in thesample is compared to an average expression level of the same miRNA in areference sample or profile. Where the reference standard or profile isfrom one or more subjects with the NSCLC, squamous cell carcinoma,wherein an increase in the subject level from the reference level isindicative of a diagnosis of adenocarcinoma. This biomarker maysimilarly be employed in assessing therapeutic efficacy in multiplesamples from the treated subject as described above.

Thus in the diagnosis of AC from other cancerous lung diseases, themiRNA biomarker miR-328 alone or in combination with other miRNAbiomarkers in a profile is useful in diagnostic methods.

In another aspect, a method of diagnosing or detecting SCC in amammalian subject is provided, comprising detecting in a biologicalsample of the subject, e.g., whole blood or peripheral blood mononuclearcells (PBMC), or from an miRNA expression profile generated from thesample, the expression level of the miRNA nucleic acid sequenceshsa-miR-328. The miRNA expression level of the specified miRNA in thesample is compared to an average expression level of the same miRNA in areference sample or profile. Where the reference standard or profile isfrom one or more subjects who are healthy and have never smoked, adecrease in the subject level from the reference level is indicative ofa diagnosis of SCC. This biomarker may similarly be employed inassessing therapeutic efficacy in multiple samples from the treatedsubject as described above.

Thus in the diagnosis or detection of SCC, the miRNA biomarker miR-328alone or in combination with other miRNA biomarkers in a profile isuseful in diagnostic methods.

Still other similar methods are contemplated based upon other miRNAidentified in Table 1 and for which the fold expression between twopopulations is useful in distinguishing COPD from NSCLC, in distinguishbetween the NSCLC stages and types and indistinguishing subjects withbenign nodules or other lung lesions related to smoking from a specifiedlung disease.

B. Methods of Diagnosing or Detecting COPD

In another aspect, a method of diagnosing or detecting chronicobstructive pulmonary disease (COPD) in a mammalian subject involvesdetecting in a sample of the subject, e.g., whole blood or peripheralblood mononuclear cells (PBMC), or from an miRNA expression profilegenerated from the sample, the expression level of the miRNA nucleicacid sequences hsa-miR-let-7d. The miRNA expression level of thespecified miRNA in the sample is compared to an average expression levelof the same miRNA in a reference sample or profile. Where the referencestandard is a standard or profile derived from the PBMC of a referencehuman subject, or an average of multiple subjects, who have benign lungnodules, an increase in expression of the miRNA from that in thereference is indicative of COPD. Where the reference standard or profileis from one or more healthy subjects who have never smoked, an increasein expression of the miRNA from that in the reference is indicative ofCOPD. Where the reference standard or profile is from one or more formeror current smokers who have no disease, an increase in expression of themiRNA from that in the reference indicates a diagnosis of COPD. Wherethe reference standard or profile is from one or more subjects diagnosedwith AC, an increase in expression of the miRNA from that in thereference indicates a diagnosis of COPD. Where the reference standard orprofile is from one or more subjects diagnosed with SCC, an increase inexpression of the miRNA from that in the reference indicates a diagnosisof COPD. This biomarker may similarly be employed in assessingtherapeutic efficacy in multiple samples from the treated subject asdescribed above.

In another aspect, a method of diagnosing or detecting chronicobstructive pulmonary disease (COPD) in a mammalian subject is provided,comprising detecting in a sample, e.g., the subject's whole blood orperipheral blood mononuclear cells (PBMC), or from an miRNA expressionprofile generated from the sample, the expression level of the miRNAnucleic acid sequences hsa-miR-221. The miRNA expression level of thespecified miRNA in the sample is compared to an average expression levelof the same miRNA in a reference sample or profile. Where the referencestandard is a standard or profile derived from the PBMC of a referencehuman subject, or an average of multiple subjects, who have benign lungnodules, an increase in expression of the miRNA from that in thereference is indicative of COPD. Where the reference standard or profileis from one or more healthy subjects who have never smoked, an increasein expression of the miRNA from that in the reference is indicative ofCOPD. Where the reference standard or profile is from one or more formeror current smokers who have no disease, an increase in expression of themiRNA from that in the reference indicates a diagnosis of COPD. Wherethe reference standard or profile is from one or more subjects diagnosedwith AC, an increase in expression of the miRNA from that in thereference indicates a diagnosis of COPD. Where the reference standard orprofile is from one or more subjects diagnosed with SCC, an increase inexpression of the miRNA from that in the reference indicates a diagnosisof COPD. This biomarker may similarly be employed in assessingtherapeutic efficacy in multiple samples from the treated subject asdescribed above.

Thus in the diagnosis of COPD from lung cancer, one or both the miRNAbiomarkers miR-let-7d or miR-221 alone or in combination with othermiRNA biomarkers in a profile is useful in diagnostic methods.

C. Method of Monitoring Post-Surgery Subjects

In another aspect, a method of diagnosing or detecting an NSCLC orrecurrence of same, or a method for monitoring the progress ofpost-surgery patients is provided. The post-surgery patients are thosewho have had an NSCLC tumor removed from the lungs. Such patients may bemonitored for recurrence or remission following surgery by detecting ina sample, e.g., the subject's whole blood or peripheral bloodmononuclear cells (PBMC), or from an miRNA expression profile generatedfrom the sample, the expression level of one or more of the miRNAnucleic acid sequences hsa-miR-let-7c, hsa-miR-34a, hsa-miR-202,hsa-miR-769-5p, and hsa-miR-642. The miRNA expression level of the oneor more specified miRNA in the sample is compared to an averageexpression level of the same miRNA in a reference sample or profile.Where the reference standard is a standard or profile derived from thebiological sample of the same subject before surgery, or an average ofmultiple subjects, who have not had surgery for tumor removal, a changein expression of the miRNA between post- and pre-surgery can indicatewhether the cancer is recurring or remitting. See, e.g., Example 3.These biomarkers or a pattern thereof may be employed in assessingtherapeutic efficacy in multiple samples from the treated subject asdescribed above.

Thus in the monitoring of post-surgery samples from lung cancersubjects, one or both the miRNA biomarkers identified above, incombination with other miRNA biomarkers in a profile is useful tomonitor patient progress.

Still other similar methods are contemplated based upon other miRNAidentified in Table 1 and for which the fold expression between twopopulations is useful in distinguishing COPD from NSCLC, in distinguishbetween the NSCLC stages and types and indistinguishing subjects withbenign nodules or other lung lesions related to smoking from a specifiedlung disease.

The diagnostic compositions and methods described herein provide avariety of advantages over current diagnostic methods. Among suchadvantages are the following. As exemplified herein, subjects withadenocarcinoma or squamous cell carcinoma of the lung, the two mostcommon types of lung cancer are distinguished from subjects withnon-malignant lung diseases including chronic obstructive lung disease(COPD) or granuloma or other benign tumors. These methods andcompositions provide a solution to the practical diagnostic problem ofwhether a patient who presents at a lung clinic with a small nodule hasmalignant disease. Patients with an intermediate-risk nodule wouldclearly benefit from a non-invasive test that would move the patientinto either a very low-likelihood or a very high-likelihood category ofdisease risk. An accurate estimate of malignancy based on a miRNAprofile (i.e. estimating a given patient has a 90% probability of havingcancer versus estimating the patient has only a 5% chance of havingcancer) would result in fewer surgeries for benign disease, more earlystage tumors removed at a curable stage, fewer follow-up CT scans, andreduction of the significant psychological costs of worrying about anodule. The economic impact would also likely be significant, such asreducing the current estimated cost of additional health care associatedwith CT screening for lung cancer, i.e., $116,000 per quality adjustedlife-year gained. A non-invasive PBMC miRNA test that has a sufficientsensitivity and specificity would significantly alter the post-testprobability of malignancy and thus, the subsequent clinical care.

A desirable advantage of these methods over existing methods is thatthey are able to characterize the disease state from aminimally-invasive procedure, i.e., by taking a blood sample. They arealso able to be performed on subjects having very small tumors in whicha biopsy would be problematic or on subjects in whom no tumor is knownor visible. Blood samples have an additional advantage, which is thatthe material is easily prepared and stabilized for later analysis. Thus,the methods and compositions described herein could prevent patientsfrom undergoing unnecessary procedures (i.e. if a small lung nodule isdiscovered) or potential be used to screen high risk patients. Themethods and compositions described herein may also be useful in otherpopulations, i.e., to screen certain high-risk lung cancer populations,such as asbestos exposed smokers. In yet another embodiment, the methodsand compositions described herein may be used in conjunction withclinical risk factors to help physicians make more accurate decisionsabout how to manage patients with lung nodules.

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations that become evident as a result of the teaching providedherein.

As disclosed below in the examples, miRNA expression patterns for themiRNAs; miR148a, miR221, and miR142-5p are discovered to be useful todistinguish early non-small cell lung cancer from an at risk controlgroup of chronic obstructive pulmonary diseases with similar smokinghistories. miR-221 was observed to decline in blood of early non-smallcell lung cancer patients. The increased miR-221 levels in peripheralblood mononuclear cells of chronic obstructive pulmonary diseasepatients compared to early non-small cell lung cancer and non-diseasedcontrols likely reflects impaired proliferation and accelerateddifferentiation of bronchoepithelial cells via kit regulatory cell cyclemechanism, possibly predisposing cells for malignancy. Significantlylower miR-142-5p levels were found in blood of early adenocarcinomapatients compared to diseased (chronic obstructive pulmonary disease)and non-diseased (smoker, never smoker) controls. Let-7d expression wassignificantly lower in stage SCC patients compared to chronicobstructive pulmonary disease. Interestingly, no difference was found inperipheral blood miR-155 expression between non-small cell lung cancerand chronic obstructive pulmonary disease (data not shown), although itis anticipated to be useful in distinguishing between other populations.

Example 1: Changes in microRNA Expression in PBMC Associated with EarlyStage NSCLC—Isolation and Taqman® Low Density Array Assays

Lung cancer and chronic obstructive pulmonary disease patient bloodsamples were collected and blood samples from tobacco smokers and neversmokers were collected bya phlebotomist. Peripheral blood mononuclearcells were isolated and total RNA prepared as previously described(Nebozhyn M, et al. 2006 Blood; 107(8):3189-96; Kari L, et al. 2003 J.Exp. Med., 197(11):1477-88) using TriReagent (Sigma). RNA quality andquantity were determined by BioAnalyzer 2100 (Agilent Technologies) andNanoDrop (Thermo Fisher), respectively. The lung adenocarcinoma (AC-E)and squamous cell carcinoma (SCC-E) samples were from metastasis freestage I and II non-small cell lung cancer patients. The control groupscomprised primarily smokers and ex-smokers diagnosed with chronicobstructive pulmonary disease (COPD), current and past smokers withoutdiagnosed chronic airway inflammation (S) and never smokers (NS).

The ABI TLDA, a quantitative RealTime PCR (qRT-PCR) platform that allowssimultaneous detection of 365 mature human miRNAs (ABI cataloguenumber), was used to assay miRNA expression in RNA pools derived frompatient and control groups. Multi-plexed miRNA-specific stem-loop RTprimers (ABI) provide the specificity for distinguishing closely relatedmiRNAs from less than 1 ug total RNA without prior small RNApurification making the TLDA platform particularly useful for screeningclinical samples. All TaqMan® Low Density Arrays (TLDA) were assayedwith RNA pools of 6 individuals to identiy candidate differentiallyexpressed miRNAs. The TaqMan® miRNA reverse transcription kit with HumanPanel RT primer pool v1.0 (8 pools of 48 miRNA primer sets) was used forcDNA synthesis as directed (ABI). RT reactions were carried out using 50ng total RNA for each RT primer pool, except where noted (recommendedamount 10-100 ng).

TLDA data normalization was carried out using RQ Manager software v1.2.After baseline and threshold adjustments by individual miRNAs in RQManager, the data was exported in tab-delimited format and processedfurther using Matlab 6.5 functions. Ct values were adjusted using thecorresponding fluorescence threshold of 0.2 according to the formula:Ct=RQ.Ct+log₂(0.2/RQ.Xt), where both RQ.Ct (cycle threshold value) andRQ.Xt (fluorescence threshold value) were determined by RQ Manager. Inorder to avoid switching from one calibrator sample to another whileusing the delta-delta Ct algorithm, the adjusted Ct values wereconverted to absolute expression (AE) values according to the formula:AE=0.2/2^(Ct), where 0.2 is ABI's default fluorescence threshold value.

To correct for loading differences, the AE value of each miRNA wasnormalized to 2 endogenous controls, small nucleolar RNAs (RNU44 andRNU48) previously tested to exhibit consistent expression levels acrossa large number of different tissues and cell lines (Liang Y, et al, 2007BMC Genomics 8:166). Both controls showed highly correlated expressionacross our samples (Spearman r=0.96) and the average expression valuebetween RNU44 and RNU48 was used as the normalization coefficient.

TaqMan® Low Density Array data filtering for hierarch clustering wasaccomplished as follows. Normalized AE data including floored lowexpression values were filtered to remove miRNAs that did not show foldchange (fc) of 2 or more between at least two of the sample pools.Hierarchical clustering was carried out using normalized Euclideandistance for samples and correlation similarity metric for miRNAs.Hierarchical clustering pseudocolor scale represented the log2-transformed ratio of each miRNA expression value over its averageexpression across all samples. Matlab 6.5 functions (mostly fromBioinformatics Toolbox) were used for the analysis.

TLDA results were validated with individual TaqMan assays. TaqMan® miRNAreverse transcription kit (Applied Biosy stems) was used as follows.Each 15 ul RT reaction contained 100 ng total RNA, 1×RT buffer, 1 mMdNTP mix, 50 U MultiScribe™ reverse transcriptase, 3.76 U RNaseinhibitor, and the appropriate 1× Human multiplex RT primer pool (as forTLDA). RT product was then diluted 3-fold for individual TaqMan® miRNAassay. Each 10 μl PCR reaction contained 1.33 μl of diluted RT reaction,5 μl of 2× TaqMan® Universal PCR mix (Applied Biosystems) and 0.5 μl of20× individual TaqMan® miRNA assay (Applied Biosystems). The reactionswere assembled in 384-well plates in triplicate in the ABI 7900HT,heated at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 secand 60° C. for 60 sec. Semi-automated multi-well distribution of sampleswas done using electronic multi-channel pipettes (Matrix Technologies,Hudson, N.H.). Data was normalized and processed in a same manner as forTLDA. RNU44 and RNU48 controls were run separately and AE values usedfor normalization of miRNA.

The correlation coefficient (r) between individual TaqMan® assays wascalculated using Pearson regression. Comparisons of miRNA expressionbetween individual patient samples (n=9-10 per group) were done usingMann-Whitney U test.

Raw intensities and detection p-values were extracted using IlluminaBead Studio v3.0. Arrays were quantile normalized and average backgroundwas subtracted from expression values. Non-informative probes wereremoved if their intensity was low relative to background in majority ofsamples or if maximum ratio between any 2 samples was not at least 1.2.

Lists of targets for hsa-let-7a, hsa-let-7d, hsa-miR-142-5p,hsa-miR-148a, hsa-miR-221 and hsa-miR-328 were predicted by miRandaalgorithm and retrieved from Memorial Sloan-Kettering Cancer Centerdatabase http://www.microrna.org (REF: Betel D, Wilson M, Gabow A, MarksD S, Sander C., The microRNA.org resource: targets and expression.Nucleic Acids Res. 2008 January). Expression levels for genes from eachlist of predicted targets were correlated with expression levels ofcorresponding miRNA, using data points from 33 individual samples (10AC, 11LSCC, 12 COPD), and only genes with significant negativecorrelation (p-value<0.05) were retained for further functionalanalysis. Ingenuity IPA software Core Analysis was performed to findsignificantly enriched canonical pathways and biological functions foreach group of potential direct target candidates.

Results were reported as TLDA performance assessment: reproducibility,fold change detection and input RNA. The technical variability betweenindividual TLDAs and the maximum reliable Ct cut off was empiricallyassessed as follows. One RNA pool (n=6) was assayed on 3 TLDAs using thesame RT product. Two TLDAs were run on the same day and then a 3^(rd)TLDA was run one month later to assess RT stability. Data for the threetechnical replicates were analyzed using Spearman rank correlation (r)on Ct values for individual miRNAs. The average correlation between thereplicates was calculated for three possible pair-combinations: run 1vs. run 2, run 2 vs. run 3, and run 1 vs. run 3.

Based on Ct values less than 39 the overall correlation between the 3technical replicates was r=0.98 (FIG. 1A), similar to the correlationbetween technical replicates using the single TaqMan® assays which was0.998 for 6 selected miRNAs (data not shown). The high correlationbetween samples run 1 month apart (run 3) also indicated that thediluted RT product is relatively stable for at least a month when storedat −20° C. The correlations between the 3 replicas begin to decreasebetween Ct 31-32, indicating the data is less reliable at Ct cutoffsbeyond this point. This cutoff is significantly lower then what isusually accepted for single TaqMan® assays. In order to further definethe point where reliability decreases, a ‘sliding’ correlation analysiswas used. The sliding window consisting of 40 miRNAs with centered Ctvalue from 28 to 37.5 was used to calculate average Spearman correlationcoefficient between 3 technical replicates based on Ct values for 40miRNAs. The critical point where the correlations between replicatesfell below 0.9 was found to be Ct 31.2. Moreover, at Ct 32 it hasdropped to 0.8 (FIG. 1B).

Based on these studies, a Ct value of 31 was used as the upper level cutoff for reliable TLDA data. To avoid generating unreliable fold changesfor low expressed values, all normalized AE values above the defined Ctcut off of 31, were adjusted to a defined AE value. This flooring level(flooring level) was defined as the minimal normalized expression levelacross all samples for miRNAs with unreliable Ct values above Ct 31.Flooring level was determined from normalized absolute expression values(AE_(norm)) for miRNAs with Ct value greater than cut off as follows:

${FL} = {\min\limits_{C_{t} \leq {cutoff}}\left( {AE}_{norm} \right)}$

All normalized AE values that were less than the flooring level were setequal to flooring level to calculate miRNA fold changes and insure nooverestimation of expression differences.

In addition to using TLDA technical replicates, % false positive (falsepositive AE−) miRNAs detected within specific Ct ranges beginning cycle19 up to cycle 39 were used (Table 2).

TABLE 2 Summary of fold change detection with corresponding falsepositive rates by various Ct range in three early stage adenocarcinomatechnical replicates run on three separate TLDAs. Ct Number of Replicatefold % false positive AE- range detected miRNAs change max 1.5-fold2-fold 19-20 1 1.18 0% 0% 20-21 0 NA 0% 0% 21-22 3 1.27 0% 0% 22-23 0 NA0% 0% 23-24 5 1.18 0% 0% 24-25 9 1.23 0% 0% 25-26 11 1.20 0% 0% 26-27 91.21 0% 0% 27-28 8 1.42 0% 0% 28-29 14 1.40 0% 0% 29-30 16 1.52 6% 0%30-31 11 1.45 0% 0% 31-32 14 2.42 29%  14%  32-33 17 3.47 65%  41% 33-34 10 2.58 70%  20%  34-35 13 4.77 92%  85%  35-36 12 5.33 92%  83% 36-37 6 9.76 100%  100%  37-38 5 7.65 100%  100%  38-39 10 46.43  90% 90% Abbreviations used: fold change max=The largest actual fold changedetected between triplicate TLDAs; false positive AE−=false positive;NA—not applicable.

Fold changes (1.5- and 2-fold changes) can be reliably detected with0-6% false positive AE− up to cycle 31, while % false positive AE−sharply increases above Ct 31.

To determine whether more miRNAs could be detected if input RNA wasincreased up to 100 ng as the recommended range is 10-100 ng per RT, thesame early stage squamous cell carcinoma pooled peripheral bloodmononuclear cells sample was run on TLDA with 50 and 100 ng input RNAand miRNA expression was compared. Twenty-six (26) more miRNAs could bedetected in 100 ng peripheral blood mononuclear cells pool, and thenormalized cycle difference, which was calculated as RNU-corrected cycledifference per miRNA between 50 and 100 ng samples, increasedtremendously with PCR cycle miRNA.

Example 2: miRNA Expression in Pooled RNA Samples

To determine whether differences in miRNA expression could be also bedetected in peripheral blood mononuclear cells RNA from surgicalcandidates with early stage non-small cell lung cancer as compared topatients with smoking-related chronic obstructive pulmonary disease,smokers and never smokers, pooled RNAs from patients (all ex-smokers)with stage I+II lung adenocarcinoma (early stage adenocarcinoma) or lungsquamous cell carcinoma (early stage squamous cell carcinoma) wereanalyzed with three different control groups (chronic obstructivepulmonary disease, S, NS) on the TLDAs. Each pooled RNA consisted of 6individuals described in Table 3.

TABLE 3 Clinical and demographic profiles of non-small cell lung cancerpatients and controls used on TLDAs. Smoking History TLDA PBMC GenderRace Pack- Yrs tobacco- State pool (n = 6) M/F W Other Age* Years*free** T N Mt early stage AC 2/4 5 1 67 45 1 ÷ 41 2 2 0 early stage SCC3/3 5 1 73 61 1 ÷ 26 2 2 0 COPD 2/4 2 4 64 57 4 ÷ 18 0 0 0 Smoker 1/5 24 49 22 NA 0 0 0 Non-smoker 5/1 2 4 48 0 NA 0 0 0 Abbreviations used:non-small cell lung cancer - non-small cell lung cancer; early stageadenocarcinoma - early (stage I + II) adenocarcinoma; early stagesquamous cell carcinoma - early (stage I + II) squamous cell carcinoma;chronic obstructive pulmonary disease - chronic obstructive pulmonarydisease; S—smoker; NS—never smoker; F—female; M—male; W—white; T—tumor;N—lymph node; Mt—metastasis; NA—not applicable; *average is shown;**range (min ÷ max) is shown.

It was determined that 268 out of 365 miRNAs on the array were notexpressed at detectable levels (Ct<31) in any of the 6 RNA pools tested.Seventy eight miRNAs were detected in all pooled peripheral bloodmononuclear cells samples, although not necessarily at the same levels.A total of 43 miRNAs were found to change 2-fold or more between any twoof the pooled samples. Clustering of the 43 miRNAs (Table 1 and FIG. 3)showed that the S and NS pools were more closely related and the chronicobstructive pulmonary disease and early stage squamous cell carcinomasamples were most different among all the samples. Pooled peripheralblood mononuclear cells RNA from patients with late stage (III+IV)non-small cell lung cancer exhibited an even higher number than chronicobstructive pulmonary disease of miRNAs detected (total 104, not shown),and was used for control purposes only.

To determine the miRNA candidates to be validated on individual assays,the level of change in expression that could be detected with goodaccuracy and sensitivity was assessed. As reported above, fold changesof 1.5 fold or greater could be detected on the TLDA with a falsepositive rate less than 6% for Ct values below 31 but increased to 30%for Ct between 31 and 32. To minimize false positives the criteria fordifferential expression was set at a fold change 2.0 or greater and Ctbelow 31. Out of the 43 miRNAs differentially expressed between any 2samples (FIG. 5), 6 miRNAs (let-7a, let-7d, miR-142-5p, miR-148a,miR-221, and miR-328) were identified that had Ct values<31 and foldchange>2 (Table 4). The expected false positive AE− rate at these foldchange and Ct parameters is <10%.

TABLE 4 miRNA candidates selected on TLDA validated using singleTaqMan ® miRNA assays. More than 2-fold change in at least onecomparison with NSCLC COPD vs. COPD vs. miRNA early stage AC early stageSCC let-7a 1.99 2.66 let-7d 1.95 3.02 miR-142-5p 3.38 7.0 miR-221 2.344.42 miR-148a 1.92 3.55 miR-328 2.31 4.96 Abbreviations used:COPD—chronic obstructive pulmonary disease; NSCLC—non-small cell lungcancer; early stage AC—early (stage I + II) adenocarcinoma; early stageSCC—early (stage I + II) squamous cell carcinoma.

To validate miRNAs selected on RNA pools on new samples, single TLDAmiRNA assays were conducted for the 6 miRNAs of Table 4 using RNA from10 or more individuals per group—patients and controls—that were notincluded in the peripheral blood mononuclear cells pools assayed on theprevious TLDAs. In addition the same miRNA candidates on 10 samples fromcurrent and never smokers were analyzed.

Four (let-7a, let-7d, miR-221, miR-148a) out of the six miRNAs testedwere significantly different in individual patient samples for chronicobstructive pulmonary disease vs. early stage squamous cell carcinomaand two (let-7a, miR-142-5p) miRNAs for chronic obstructive pulmonarydisease vs. early stage adenocarcinoma (Table 5).

P values were calculated using Mann-Whitney U test. Pooled groupscontained combined total RNA from 6 individuals each and were run onTLDA. Individual mixed (Ind. Mixed) groups contained 10 patients partlynew and partly from TLDA pool (see text for details). Individual new(Ind. New) groups contained 10 completely new patient samples. See FIGS.4A-4D. When expression of the candidate miRNAs in the independentsamples from chronic obstructive pulmonary disease, AC and LSCC patientswere compared by single TaqMan® assays (Table 5), the direction ofchange in the individual assays was the same for all 6 samples, althoughratios are lower than detected on the TLDAs. For 3 out 6 candidatemiRNAs (miR-221, miR-142-5p and miR-148a) the result was statisticallysignificant in one of the 2 comparisons. The normalized expression of 4significant miRNA candidates tested on all five groups including smokers(S) and never smokers (NS) are shown in FIGS. 4A-4D. Although let7d wasnot informative for the comparison of chronic obstructive pulmonarydisease with non-small cell lung cancer, it is significantly differentbetween chronic obstructive pulmonary disease and “healthy” smokers aswell as never smokers.

Moreover, with 100% new individual patient samples miR-148a became astronger candidate allowing differentiation between 2 early non-smallcell lung cancer types: adenocarcinoma and squamous cell carcinoma. Inaddition, let-7d and miR-221 separated well chronic obstructivepulmonary disease (mainly former smokers) from either current smokers(S) and/or never smokers (NS), and miR-142-5p was expressed atsignificantly lower levels in early adenocarcinoma (early stageadenocarcinoma) patients compared to diseased and non-diseased controls:chronic obstructive pulmonary disease, smoker and non smoker. Eventhough TLDA-derived miR-142-5p expression was not confirmed on 100% newearly stage squamous cell carcinoma individual patient samples, therewas a tendency to follow the early stage adenocarcinoma pattern.MiR-142-5p was expressed at significantly lower levels in AC-E patientscompared to all control groups COPD, S and NS as was found with thepooled comparisons. However the high differential expression detectedbetween the pooled COPD and SCC samples was not confirmed by theindividual assays. There was a general tendency of higher expression inthe COPD samples but the difference did not reach statisticalsignificance.

Additional studies are ongoing that use a single reaction mega-plex RTprimer pool (450 miRNAs) and a newly developed miRNA pre-amplificationthat permit studies with small amounts of RNA from highly limitedclinical samples.

Example 3: Differential Expression of miRNAs in NSCLC Patients afterTumor Removal

There is a complex of direct-mRNA/TF upstream/miRNA regulationsimplicated by the presence of cancer (or tumor) present. A strongsignature of cancer is observed in blood immune cells, which mostlydownregulates immune functions. Multiple miRNAs target key nodes ofimmune processes. There is a trend of upregulation of miRNAs inbiological, e.g., PBMC, samples of subjects with NSCLC before surgicalresection of tumor tissue (PRE-) and after surgery (POST-). Specifically5 miRNAs are highly significantly changed.

MicroRNA transcription was analyzed on a subset of patient samples,using Illumina bead arrays. PBMC from the 18 patient pairs werecollected. Of these 18 patients, 10 were diagnosed with adenocarcinoma,6 were diagnosed with LSCC and 2 were diagnosed as unclassified NSCLC.Times of sample collection post-surgery ranged from 1 to 5 months withmajority of samples (10) being taken 2 months after surgery and only 2samples taken at 1, 3, 4 and 5 months post-surgery. All samples werecollected before any additional therapy was started. Blood samples weredrawn in two “CPT” tubes (BD). PBMC were isolated within 90 minutes ofblood draw, washed in PBS, transferred into RNA Later (Ambion) and thenstored at 4° C. overnight before transfer to −80° C. Extracted RNA wasused for further processing.

Illumina microRNA expression profiling platform was used to studychanges in expression levels of microRNAs in PBMC taken post surgery in11 of 18 patients. RNA purification was carried out using TriReagentMolecular Research) as recommended and controlled for quality using theBioanalyzer. Only samples with 28S/16S ratios >0.75 were used forfurther studies. A constant amount (500 ng) of total RNA was amplifiedas recommended by Illumina. Samples were hybridized to the humanIllumina MicroRNA v2 (Universal 12 Beadchip 1536 bead type) SentrixBeadchip Array. Illumina BeadStudio v.3.0 software was used to exportexpression levels and detection p-values for each probe of each sample.Arrays were normalized to 95^(th) percentile of overall slide expressionand filtered to remove non-informative miRNA probes. A probe was callednon-informative if it had expression signals with detection p-value>0.05in all samples or if it had expression signals less than 2 averagebackground levels in all samples.

miRNA expression data for 11 pre/post pairs of samples was tested fordifferential expression using two-tail pairwise t-test with significanceset to p-value<0.05 unless stated otherwise. False Discovery Rate wascalculated according to Storey JD procedure (Storey Tibshirani 2003).SVM-RFE. List of ranked genes was received using linear kernel SVM-RFE²²with 10-fold 10 resampling cross validation. Each cross-validationiteration started with 1000 top significant by t-test genes and thenumber of genes was reduced by 10% at each feature elimination stepbased on gene SVM-scores. Final ranking of the genes was done by Bordacount procedure. Heatmaps. Heatmap for a list of genes is composed using2-way hierarchical clustering using Euclidean distance to clustersamples/conditions and Spearman correlation distance to cluster genes.Pathway and functional analysis was carried out with Ingenuity PathwaysAnalysis software using Ingenuity Core Analysis (IPA 6.0, Ingenuity®Systems) with Benjamini-Hochberg multiple testing corrected p-value<0.05as a significance threshold.

Enrichments of Gene Ontology (GO) terms, KEGG and BIOCARTA pathwaysalong with Swiss-Prot, INTERPRO and SMART keywords in a gene list wasdone with DAVID software. Results were filtered to satisfy FDR<5% andFold Enrichment>1.5 criteria. Putative miRNA target genes.Computationally predicted target genes for a miRNA were derived fromresults of miRanda target scanner software runs as provided by Sanger(United Kingdom; website indicated by microrna.sanger.ac.uk) orSloan-Kettering (website indicated by microrna.org) databases. Overlapwith gene expression data was done using Entrez Gene IDs and acomputationally predicted target gene was called a putative miRNA targetif it was significantly downregulated in PRE surgery samples as assessedby one-tail paired t-test with significance threshold p-value<0.1.

Q-PCR validation of array results was carried out using the ABI TaqManSystem as recommended, in an ABI 7900HT PCR System. Each sample wasanalyzed in duplicate and samples with CVs between replicates that weremore than 0.5 delta Ct were repeated.

From 1146 expressed probes on the array, 643 differentially expressedmiRNAs met the selection criteria of being expressed in at least onesample. Of the 643 miRNAs detected, 108 were putative miRNAs that werepredicted by parallel sequencing. The rest of the detected probestargeted 443 unique miRNAs with 92 minor miR* forms. Forty-six (46)known miRNAs (paired t-test, p-value<0.05) were differentially expressedbetween pre- and post-surgery samples (FDR of 42%). Out of those 46miRNAs, 42 (91%) showed upregulation in samples taken pre surgery.

Universally altered expression was detected of five miRNAs let-7c,miR-34a, miR-202 in its minor form (hsa-miR-202*) and miR-769-5p in thepre compared to the post-surgery samples. These five miRNAs satisfiedthe criteria of a p-value<0.002 and a FDR<15%. All were upregulated inpre-surgery samples. Magnitudes of the changes in each patient in thosemiRNAs are presented in FIG. 8. As can be seen from the figure, let-7cis shows upregulation in all 11 tested patients with median upregulationof 33% and a range from 4% to 52%. The other miRNAs show upregulation in10 of the 11 patients (values below zero in FIG. 3) with medianupregulation in the rest of the patients as follows: 35% for miR-34a,14% for miR-202*, 17% for miR-769-5p and 51% for miR-642. Only patients2, 4, 5 and 17, all adenocarcinomas, have one of the 5 miRNAs which doesnot follow the general trend in the other 10 samples and this miRNA wasdifferent for each of these patients.

These five miRNAs may have common regulators as identified bytranscription factor binding site (TFBS) analysis. These 5 miRNAs havepredicted targets involved in immune functions. It is theorized that thetargets are generally downstream pathway targets, such as kinases andtranscription factors). The Toll-like receptor (TLR) pathway issignificantly targeted by those miRNAs. Expression of these miRNAs isnegatively correlated with subset of genes that are differentiallyexpressed between pre and post surgery samples. All of these miRNAstarget downstream genes of TLR signaling pathway. The tumor presenceinduces a downregulation of TLR signaling pathways through miRNAtargeting various key molecules in these pathways.

Each and every patent, patent application, and publication, includingwebsites cited throughout the disclosure, and U.S. provisionalapplication No. 61/112,744 is expressly incorporated herein by referencein its entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention are devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims include such embodiments and equivalent variations.

1. A method of diagnosing or detecting or assessing a condition in amammalian subject comprising (a) detecting in a biological sample of thesubject, or from an miRNA expression profile generated from the sample,the expression level of an miRNA nucleic acid sequence identified inTable 1; (b) comparing the miRNA expression level or miRNA expressionprofile from the subject's sample to a reference miRNA standard; whereina change in expression of the subject's sample miRNA from that in thereference miRNA standard indicates a diagnosis or prognosis of acondition selected from the group consisting of a lung cancer, chronicobstructive pulmonary disease (COPD), and benign lung nodules.